Benzocyanine Compounds

ABSTRACT

Compounds useful as labels with properties comparable to known fluorescent compounds. The compounds are conjugated to proteins and nucleic acids for biological imaging and analysis. Synthesis of the compounds, formation and use of the conjugated compounds, and specific non-limiting examples of each are provided.

This application claims priority to co-pending U.S. Ser. No. 13/571,858filed Aug. 10, 2012 now U.S. Pat. No. 9,252,003; which claims priorityto U.S. Ser. Nos. 61/524,167 filed Aug. 16, 2011; 61/604,232 filed Feb.28, 2012; and 61/607,737 filed Mar. 7, 2012, each of which is expresslyincorporated by reference herein in its entirety.

Compounds useful as labels with properties comparable to knownfluorescent compounds are disclosed. The compounds can be conjugated toproteins and nucleic acids for biological imaging and analysis.Synthesis of the compounds, formation and use of the conjugatedcompounds, and specific non-limiting examples of each are disclosed.

Compounds that react with biomolecules (e.g., antigens, antibodies,DNA-segments with the corresponding complimentary species for measuringenzyme kinetics, receptor-ligand interactions, nucleic acidhybridization kinetics in vitro as well as in vivo, etc.), termed labelsor dyes, are useful for, e.g., pharmacological characterization ofreceptors and drugs, binding data, etc. Compounds such as xanthyliumsalts (U.S. Pat. No. 5,846,737) and/or cyanines (U.S. Pat. No.5,627,027) are used for such applications, but aggregate and formdimers, especially in aqueous solution, due to planarity of theirπ-system. Compounds that have insufficient hydrophilicity undergonon-specific interactions with various surfaces, resulting in problemswhen attempting to purify the corresponding conjugate, and anunsatisfactory signal to noise ratio.

Efforts are directed to reducing undesirable properties by introducingsubstituents that increase the hydrophilicity of the compounds. Forexample, sulfonic acid function substituents have been introduced intothe cyanine chromophore. U.S. Pat. No. 6,083,485 (Licha) and U.S. Pat.Nos. 6,977,305 and 6,974,873 (Molecular Probes) disclose cyaninecompounds having one of the common methyl groups in the 3-position ofthe terminal indole heterocycle substituted by an ω-carboxyalkylfunction, and in which the previously present (e.g. in Cy3 or Cy5)N-alkyl or N-ω-carboxyalkyl functions are replaced by N-ω-alkyl sulfonicacid functions. WO 05/044923 discloses cyanine compounds having thecommon methyl substituent in the 3-position of the terminal indoleheterocycle substituted by a N-ω-alkyl sulfonic acid function. In thesepublications, cyanine compounds having more than two sulfonic acidfunction substituents exhibited higher solubility and correspondingly alower tendency to dimer formation, in comparison to cyanine compounds(Cy3, Cy5) described in U.S. Pat. No. 5,627,027.

The disclosed benzocyanine compounds are useful as labels in optical,e.g., fluorescence optical, determination and detection methods. Thecompounds have high hydrophilicity, high molar absorbance, highphoto-stability, and high storage stability. These compounds wereexcited by monochromatic (e.g., lasers, laser diodes) or polychromatic(e.g., white light sources) light in the ultraviolet (UV), visible, andnear infrared (NIR) spectral region to generate emission of fluorescencelight.

Typical application methods are based on the reaction of the compoundswith biomolecules, e.g., proteins (e.g., antigens, antibodies, etc.),DNA and/or RNA segments, etc. with the corresponding complimentaryspecies. Thus, among other embodiments, the compounds are used tomeasure enzyme kinetics, receptor-ligand interactions, and nucleic acidhybridization kinetics in vitro and/or in vivo. The compounds are usedfor the pharmacological characterization of receptors and/or drugs.Applications include, but are not limited to, uses in medicine,pharmacy, biological sciences, materials sciences, environmentalcontrol, detection of organic and inorganic micro samples occurring innature, etc.

The following nomenclature is used to describe the embodiments of thecompounds having either a single PEG or multiple PEGs:

The following nomenclature is used for compounds having one ethyleneglycol or one (poly)ethylene glycol, which is always at the indole N onthe left side of the cyanine: (the first compound is explained indetail, and all other compounds follow this same nomenclature format):579 Compound 1 ((ethylene glycol) (e.g., is a 579 compound, 1 is thelength of the ethylene glycol))579 Compound 2 (diethylene glycol)579 Compound 3 (polyethylene glycol (3))579 Compound 4 (polyethylene glycol (4))579 Compound 5 (polyethylene glycol (5))579 Compound 6 (polyethylene glycol (6))679 Compound 1 (ethylene glycol)679 Compound 2 (diethylene glycol)679 Compound 3 (polyethylene glycol (3))679 Compound 4 (polyethylene glycol (4))679 Compound 5 (polyethylene glycol (5))679 Compound 6 (polyethylene glycol (6))779 Compound 1 (ethylene glycol)779 Compound 2 (diethylene glycol)779 Compound 3 (polyethylene glycol (3))779 Compound 4 (polyethylene glycol (4))779 Compound 5 (polyethylene glycol (5))779 Compound 6 (polyethylene glycol (6))The following nomenclature is used for compounds having more than oneethylene glycol or more than one (poly)ethylene glycol; i.e., one isalways at the indole N on the left side of the cyanine, and at least 1more (2 total) at a site(s) other than the indole N on the left side ofthe cyanine (the first compound is explained in detail, and all othercompounds follow this same nomenclature format):579 Compound 1/X (e.g., compound 1 is the length of the PEG at the leftportion on the cyanine; X is the total number of PEGs on the entirecompound).

579 Compound 2/X 579 Compound 3/X 579 Compound 4/X 579 Compound 5/X 579Compound 6/X 679 Compound 1/X 679 Compound 2/X 679 Compound 3/X 679Compound 4/X 679 Compound 5/X 679 Compound 6/X 779 Compound 1/X 779Compound 2/X 779 Compound 3/X 779 Compound 4/X 779 Compound 5/X 779Compound 6/X

Thus, 579, 679, and 779 compounds comprise a polymethine chain of 3 C, 5C, and 7 C atoms, respectively, the first 1-6 number refers to thelength of a PEG group on an indole N position, e.g., 1 is ethyleneglycol (PEG₁), 2 is diethylene glycol (PEG₂), 3 is polyethylene glycol(3) (PEG₃), 4 is polyethylene glycol (4) (PEG₄), 5 is polyethyleneglycol (5) (PEG₅), and 6 is polyethylene glycol (6) (PEG₆), and X refersto the number of PEG groups on the compound. For example, in oneembodiment, 679 Compound 4/4 means a PEG₄ on an indole N and a total offour PEG groups on the compound.

In one embodiment, the benzocyanine compounds have, in an N-position ofone heterocycle, an ethylene glycol group or an ethylene glycol polymer(i.e., poly(ethylene) glycol, collectively abbreviated as PEG), and theother heterocycle has, in an N-position, a function for conjugating thecompound to a biomolecule. In one embodiment, the benzocyanine compoundhas, in any position of the compound, at least one sulfo (SO₃ ⁻) and/orsulfoalkyl group. In one embodiment, the benzocyanine compound has, inany position of the compound, a sulfonamide and/or carboxamide groupcomprising an ethylene glycol group or an ethylene glycol polymer (i.e.,poly(ethylene) glycol, collectively abbreviated as PEG), either directlyor indirectly attached to the compound. Indirect attachment indicatesuse of a linker, direct attachment indicates lack of such a linker. Alinker can be any moiety.

In one embodiment, the benzocyanine compounds have, in an N-position ofone heterocycle, an ethylene glycol group or an ethylene glycol polymer(i.e., poly(ethylene) glycol, collectively abbreviated as PEG), and theother heterocycle has, in a N-position, an ethylene glycol group or anethylene glycol polymer (i.e., poly(ethylene) glycol, collectivelyabbreviated as PEG) and a function for conjugating the compound to abiomolecule. In one embodiment, the benzocyanine compounds have anethylene glycol group or an ethylene glycol polymer (i.e.,poly(ethylene) glycol, collectively abbreviated as PEG) in anotherposition of the benzocyanine compound. In one embodiment, thebenzocyanine compound has, in any position of the compound, at least onesulfo and/or sulfoalkyl group. In one embodiment, the benzocyaninecompound has, in any position of the compound, a sulfonamide and/orcarboxamide group comprising an ethylene glycol group or an ethyleneglycol polymer (i.e., poly(ethylene) glycol, collectively abbreviated asPEG), either directly or indirectly attached to the compound.

In one embodiment, the compound is

where each of R¹ and R² is the same or different and is independentlyselected from the group consisting of an aliphatic, heteroaliphatic,sulfoalkyl group, heteroaliphatic with terminal SO₃, a PEG group P—Zwhere P is selected from an ethylene glycol group, a diethylene glycolgroup, and a polyethylene glycol group, where the polyethylene glycolgroup is (CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, asulfonamide group -L-SO₂NH—P—Z, and a caboxamide group -L-CONH—P—Z, andZ is selected from H, a CH₃, a CH₃ group, an alkyl group, a heteroalkylgroup, or —CO—NHS; X is selected from the group consisting of —OH, —SH,—NH₂, —NH—NH₂, —F, —Cl, —Br, I, —NHShydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O-STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, —NR-L-NH—CO—CH2-,imidazole, azide, —NR-L-O—NH₂, and —NR-L-O—CO—NHS, where R is —H or analiphatic or heteroaliphatic group, and L is selected from the groupconsisting of a divalent linear (—(CH₂)_(t)—, t=0 to 15), crossed, orcyclic alkyl group optionally substituted by at least one oxygen atomand/or sulfur atom; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or othercation(s) needed to compensate the negative charge brought by thecyanine; m is an integer from 0 to 5 inclusive; o is an integer from 0to 12 inclusive; and n is an integer from 1 to 3 inclusive. In oneembodiment, at least one of R¹ and R² contains a PEG group.

In one embodiment, the PEG group P is selected from the group consistingof —C—C—O—C (ethylene glycol with terminal methyl),—C—C—O—C—C—O—C(diethylene glycol with terminal methyl),—C—C—O—C—C—O—C—C—O—C(polyethylene glycol (3) with terminal methyl),—C—C—O—C—C—O—C—C—O—C—C—O—C (polyethylene glycol (4) with terminalmethyl), —C—C—O—C—C—O—C—C—O—C—C—O—C—C—O—C(polyethylene glycol (5) withterminal methyl), and C—C—O—C—C—O—C—C—O—C—C—O—C—C—C—O—C—C—O—C(polyethylene glycol (6) with terminal methyl). In one embodiment, thePEG group P may be either uncapped, e.g., lack a terminal methyl, or maybe capped with an atom or group other than a methyl. In one embodiment,the PEG group P terminates with a Z group, where Z is selected from H,CH₃, a CH₃ group, an alkyl group, or a heteroalkyl group.

In one embodiment, the compound is general formula Ia or general formulaIb, collectively referred to as general formula I, where R1 is methyland R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is0; o is 3; and n is 1. In one embodiment, the compound is generalformula I, where R1 is methyl and R2 is sulfoalkyl; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 1. In oneembodiment, the compound is general formula I, where R1 is methyl and R2is sulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is3; and n is 1. In one embodiment, the compound is general formula I,where R1 is methyl and R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 1. In one embodiment, thecompound is general formula I, where R1 is methyl and R2 is sulfoalkyl;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 1.In one embodiment, the compound is general formula I, where R1 is methyland R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is5; o is 3; and n is 1.

In one embodiment, the compound is general formula I, where R1 is methyland R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is0; o is 3; and n is 2. In one embodiment, the compound is generalformula I, where R1 is methyl and R2 is sulfoalkyl; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 2. In oneembodiment, the compound is general formula I, where R1 is methyl and R2is sulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is3; and n is 2. In one embodiment, the compound is general formula I,where R1 is methyl and R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 2. In one embodiment, thecompound is general formula I, where R1 is methyl and R2 is sulfoalkyl;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 2.In one embodiment, the compound is general formula I, where R1 is methyland R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is5; o is 3; and n is 2.

In one embodiment, the compound is general formula I, where R1 is methyland R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is0; o is 3; and n is 3. In one embodiment, the compound is generalformula I, where R1 is methyl and R2 is sulfoalkyl; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 3. In oneembodiment, the compound is general formula I, where R1 is methyl and R2is sulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is3; and n is 3. In one embodiment, the compound is general formula I,where R1 is methyl and R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 3. In one embodiment, thecompound is general formula I, where R1 is methyl and R2 is sulfoalkyl;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 3.In one embodiment, the compound is general formula I, where R1 is methyland R2 is sulfoalkyl; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is5; o is 3; and n is 3.

In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 1. In one embodiment, thecompound is general formula I, where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3; and nis 1. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 2; o is 3; and n is 1. In one embodiment, thecompound is general formula I, where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and nis 1. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; o is 3; and n is 1. In one embodiment, thecompound is general formula I, where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and nis 1.

In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 2. In one embodiment, thecompound is general formula I, where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3; and nis 2. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 2; o is 3; and n is 2. In one embodiment, thecompound is general formula I, where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and nis 2. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; o is 3; and n is 2. In one embodiment, thecompound is general formula I, where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and nis 2.

In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 3. In one embodiment, thecompound is general formula I, where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3; and nis 3. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 2; o is 3; and n is 3. In one embodiment, thecompound is general formula I, where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and nis 3. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; o is 3; and n is 3. In one embodiment, thecompound is general formula I, where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and nis 3.

In one embodiment, the compound is general formula I, where R1 is methyland R2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is0; o is 3; and n is 1. In one embodiment, the compound is generalformula I, where R1 is methyl and R2 is a PEG group; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 1. In oneembodiment, the compound is general formula I, where R1 is methyl and R2is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is3; and n is 1. In one embodiment, the compound is general formula I,where R1 is methyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 1. In one embodiment, thecompound is general formula I, where R1 is methyl and R2 is a PEG group;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 1.In one embodiment, the compound is general formula I, where R1 is methyland R2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is5; o is 3; and n is 1.

In one embodiment, the compound is general formula I, where R1 is methyland R2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is0; o is 3; and n is 2. In one embodiment, the compound is generalformula I, where R1 is methyl and R2 is a PEG group; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 2. In oneembodiment, the compound is general formula I, where R1 is methyl and R2is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is3; and n is 2. In one embodiment, the compound is general formula I,where R1 is methyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 2. In one embodiment, thecompound is general formula I, where R1 is methyl and R2 is a PEG group;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 2.In one embodiment, the compound is general formula I, where R1 is methyland R2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is5; o is 3; and n is 2.

In one embodiment, the compound is general formula I, where R1 is methyland R2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is0; o is 3; and n is 3. In one embodiment, the compound is generalformula I, where R1 is methyl and R2 is a PEG group; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 3. In oneembodiment, the compound is general formula I, where R1 is methyl and R2is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is3; and n is 3. In one embodiment, the compound is general formula I,where R1 is methyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 3. In one embodiment, thecompound is general formula I, where R1 is methyl and R2 is a PEG group;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 3.In one embodiment, the compound is general formula I, where R1 is methyland R2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is5; o is 3; and n is 3.

In one embodiment, the compound is general formula I, where each of R1and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is0; o is 3; and n is 1. In one embodiment, the compound is generalformula I, where each of R1 and R2 is a PEG group; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 1. In oneembodiment, the compound is general formula I, where each of R1 and R2is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is3; and n is 1. In one embodiment, the compound is general formula I,where each of R1 and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 1. In one embodiment, thecompound is general formula I, where each of R1 and R2 is a PEG group; Xis —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 1. Inone embodiment, the compound is general formula I, where each of R1 andR2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; ois 3; and n is 1.

In one embodiment, the compound is general formula I, where each of R1and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is0; o is 3; and n is 2. In one embodiment, the compound is generalformula I, where each of R1 and R2 is a PEG group; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 2. In oneembodiment, the compound is general formula I, where each of R1 and R2is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is3; and n is 2. In one embodiment, the compound is general formula I,where each of R1 and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 2. In one embodiment, thecompound is general formula I, where each of R1 and R2 is a PEG group; Xis —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 2. Inone embodiment, the compound is general formula I, where each of R1 andR2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; ois 3; and n is 2.

In one embodiment, the compound is general formula I, where each of R1and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is0; o is 3; and n is 3. In one embodiment, the compound is generalformula I, where each of R1 and R2 is a PEG group; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 3. In oneembodiment, the compound is general formula I, where each of R1 and R2is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is3; and n is 3. In one embodiment, the compound is general formula I,where each of R1 and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 3. In one embodiment, thecompound is general formula I, where each of R1 and R2 is a PEG group; Xis —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 3. Inone embodiment, the compound is general formula I, where each of R1 andR2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; ois 3; and n is 3.

In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 0; o is 3; and nis 1. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3; and nis 1. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and nis 1. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and nis 1. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and nis 1. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and nis 1.

In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 0; o is 3; and nis 2. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3; and nis 2. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and nis 2. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and nis 2. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and nis 2. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and nis 2.

In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 0; o is 3; and nis 3. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3; and nis 3. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and nis 3. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and nis 3. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and nis 3. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and nis 3.

In one embodiment, the compound is

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, heteroaliphatic with terminal SO₃, aPEG group P—Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P—Z, and a caboxamide group-L-CONH—P—Z, and Z is selected from H, a CH₃ group, an alkyl group, aheteroalkyl group, or —CO—NHS; each of R⁷, R⁸, R¹¹, R¹², R¹³, and R¹⁴ isthe same or different and is independently selected from the groupconsisting of H, SO₃, a PEG group P—Z where P is selected from anethylene glycol group, a diethylene glycol group, and a polyethyleneglycol group, where the polyethylene glycol group is (CH₂CH₂O)_(s),where s is an integer from 3-6 inclusive, a sulfonamide group-L-SO₂NH—P—Z, and a caboxamide group -L-CONH—P—Z, and Z is selected fromH, a CH₃ group, an alkyl group, or a heteroalkyl group; X is selectedfrom the group consisting of —OH, —SH, —NH₂, —NH—NH₂, —F, —Cl, —Br, I,—NHS (hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O-STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, —NR-L-NH—CO—CH₂—I,imidazole, azide, —NR-L-O—NH2, and —NR-L-O—CO—NHS, where R is —H or analiphatic or heteroaliphatic group, and L is selected from the groupconsisting of a divalent linear (—(CH₂)_(t)—, t=0 to 15), crossed, orcyclic alkyl group optionally substituted by at least one oxygen atomand/or sulfur atom; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or othercation(s) needed to compensate the negative charge brought by thecyanine; m is an integer from 0 to 5 inclusive; o is an integer from 0to 12 inclusive; and n is an integer from 1 to 3 inclusive.

In one embodiment, the compound is general formulas IIa or IIb,collectively general formula II, where each of R1, R5, and R6 is methyland R2 is a PEG group; each of R7, R8, R11, and R12 is H; each of R13and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 0; ois 3; and n is 1. In one embodiment, the compound is general formula II,where each of R1, R5, and R6 is methyl and R2 is a PEG group; each ofR7, R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 1. In oneembodiment, the compound is general formula II, where each of R1, R5,and R6 is methyl and R2 is a PEG group; each of R7, R8, R11, and R12 isH; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 2; o is 3; and n is 1. In one embodiment, thecompound is general formula II, where each of R1, R5, and R6 is methyland R2 is a PEG group; each of R7, R8, R11, and R12 is H; each of R13and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; ois 3; and n is 1. In one embodiment, the compound is general formula II,where each of R1, R5, and R6 is methyl and R2 is a PEG group; each ofR7, R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 1. In oneembodiment, the compound is general formula II, where each of R1, R5,and R6 is methyl and R2 is a PEG group; each of R7, R8, R11, and R12 isH; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 5; o is 3; and n is 1.

In one embodiment, the compound is general formula II, where each of R1,R5, and R6 is methyl and R2 is a PEG group; each of R7, R8, R11, and R12is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where each of R1, R5, and R6 is methyland R2 is a PEG group; each of R7, R8, R11, and R12 is H; each of R13and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; ois 3; and n is 2. In one embodiment, the compound is general formula II,where each of R1, R5, and R6 is methyl and R2 is a PEG group; each ofR7, R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 2. In oneembodiment, the compound is general formula II, where each of R1, R5,and R6 is methyl and R2 is a PEG group; each of R7, R8, R11, and R12 isH; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where each of R1, R5, and R6 is methyland R2 is a PEG group; each of R7, R8, R11, and R12 is H; each of R13and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; ois 3; and n is 2. In one embodiment, the compound is general formula II,where each of R1, R5, and R6 is methyl and R2 is a PEG group; each ofR7, R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 2.

In one embodiment, the compound is general formula II, where each of R1,R5, and R6 is methyl and R2 is a PEG group; each of R7, R8, R11, and R12is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where each of R1, R5, and R6 is methyland R2 is a PEG group; each of R7, R8, R11, and R12 is H; each of R13and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; ois 3; and n is 3. In one embodiment, the compound is general formula II,where each of R1, R5, and R6 is methyl and R2 is a PEG group; each ofR7, R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 3. In oneembodiment, the compound is general formula II, where each of R1, R5,and R6 is methyl and R2 is a PEG group; each of R7, R8, R11, and R12 isH; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where each of R1, R5, and R6 is methyland R2 is a PEG group; each of R7, R8, R11, and R12 is H; each of R13and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; ois 3; and n is 3. In one embodiment, the compound is general formula II,where each of R1, R5, and R6 is methyl and R2 is a PEG group; each ofR7, R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 3.

In one embodiment, the compound is general formula II, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 1. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is H; each ofR13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is1; o is 3; and n is 1. In one embodiment, the compound is generalformula II, where each of R5 and R6 is methyl; each of R1 and R2 is aPEG group; each of R7, R8, R11, and R12 is H; each of R13 and R14 issulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and nis 1. In one embodiment, the compound is general formula II, where eachof R5 and R6 is methyl; each of R1 and R2 is a PEG group; each of R7,R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; o is 3; and n is 1. In oneembodiment, the compound is general formula II, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11, andR12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; o is 3; and n is 1. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is H; each ofR13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is5; o is 3; and n is 1.

In one embodiment, the compound is general formula II, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is H; each ofR13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is1; o is 3; and n is 2. In one embodiment, the compound is generalformula II, where each of R5 and R6 is methyl; each of R1 and R2 is aPEG group; each of R7, R8, R11, and R12 is H; each of R13 and R14 issulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and nis 2. In one embodiment, the compound is general formula II, where eachof R5 and R6 is methyl; each of R1 and R2 is a PEG group; each of R7,R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; o is 3; and n is 2. In oneembodiment, the compound is general formula II, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11, andR12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is H; each ofR13 and R14 is sulfo; X4 is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is5; o is 3; and n is 2.

In one embodiment, the compound is general formula II, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is H; each ofR13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is1; o is 3; and n is 3. In one embodiment, the compound is generalformula II, where each of R5 and R6 is methyl; each of R1 and R2 is aPEG group; each of R7, R8, R11, and R12 is H; each of R13 and R14 issulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and nis 3. In one embodiment, the compound is general formula II, where eachof R5 and R6 is methyl; each of R1 and R2 is a PEG group; each of R7,R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; o is 3; and n is 3. In oneembodiment, the compound is general formula II, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11, andR12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is H; each ofR13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is5; o is 3; and n is 3.

In one embodiment, the compound is general formula II, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 1. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; eachof R13 and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1;o is 3; and n is 1. In one embodiment, the compound is general formulaII, where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7, R8, R11, and R12 is sulfo; each of R13 and R14 is H; X is—OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 1. Inone embodiment, the compound is general formula II, where each of R5 andR6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11, andR12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 1. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; eachof R13 and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4;o is 3; and n is 1. In one embodiment, the compound is general formulaII, where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7, R8, R11, and R12 is sulfo; each of R13 and R14 is H; X is—OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 1.

In one embodiment, the compound is general formula II, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; eachof R13 and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1;o is 3; and n is 2. In one embodiment, the compound is general formulaII, where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7, R8, R11, and R12 is sulfo; each of R13 and R14 is H; X is—OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 2. Inone embodiment, the compound is general formula II, where each of R5 andR6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11, andR12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; eachof R13 and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4;o is 3; and n is 2. In one embodiment, the compound is general formulaII, where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7, R8, R11, and R12 is sulfo; each of R13 and R14 is H; X is—OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 2.

In one embodiment, the compound is general formula II, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; eachof R13 and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1;o is 3; and n is 3. In one embodiment, the compound is general formulaII, where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7, R8, R11, and R12 is sulfo; each of R13 and R14 is H; X is—OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 3. Inone embodiment, the compound is general formula II, where each of R5 andR6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11, andR12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; eachof R13 and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4;o is 3; and n is 3. In one embodiment, the compound is general formulaII, where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7, R8, R11, and R12 is sulfo; each of R13 and R14 is H; X is—OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 3.

In one embodiment, the compound is general formula II, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 issulfo; each of R11 and R12 is a sulfonamide group —SO₂NH—P where P is aPEG group; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 1. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a sulfonamide group —SO₂NH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is3; and n is 1. In one embodiment, the compound is general formula II,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a sulfonamide group—SO₂NH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 1. In oneembodiment, the compound is general formula II, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 is sulfo;each of R11 and R12 is a sulfonamide group —SO₂NH—P where P is a PEGgroup; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 1. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a sulfonamide group —SO₂NH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is3; and n is 1. In one embodiment, the compound is general formula II,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a sulfonamide group—SO₂NH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 1.

In one embodiment, the compound is general formula II, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 issulfo; each of R11 and R12 is a sulfonamide group —SO₂NH—P where P is aPEG group; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a sulfonamide group —SO₂NH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is3; and n is 2. In one embodiment, the compound is general formula II,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a sulfonamide group—SO₂NH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 2. In oneembodiment, the compound is general formula II, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 is sulfo;each of R11 and R12 is a sulfonamide group —SO₂NH—P where P is a PEGgroup; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a sulfonamide group —SO₂NH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is3; and n is 2. In one embodiment, the compound is general formula II,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a sulfonamide group—SO₂NH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 2.

In one embodiment, the compound is general formula II, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 issulfo; each of R11 and R12 is a sulfonamide group —SO₂NH—P where P is aPEG group; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a sulfonamide group —SO₂NH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is3; and n is 3. In one embodiment, the compound is general formula II,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a sulfonamide group—SO₂NH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 3. In oneembodiment, the compound is general formula II, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 is sulfo;each of R11 and R12 is a sulfonamide group —SO₂NH—P where P is a PEGgroup; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a sulfonamide group —SO₂NH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is3; and n is 3. In one embodiment, the compound is general formula II,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a sulfonamide group—SO₂NH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 3.

In one embodiment, the compound is general formula II, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 issulfo; each of R11 and R12 is a caboxamide group —CONH—P where P is aPEG group; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 1. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a caboxamide group —CONH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is3; and n is 1. In one embodiment, the compound is general formula II,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a caboxamide group—CONH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 1. In oneembodiment, the compound is general formula II, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 is sulfo;each of R11 and R12 is a caboxamide group —CONH—P where P is a PEGgroup; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 1. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a caboxamide group —CONH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is3; and n is 1. In one embodiment, the compound is general formula II,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a caboxamide group—CONH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 1.

In one embodiment, the compound is general formula II, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 issulfo; each of R11 and R12 is a caboxamide group —CONH—P where P is aPEG group; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a caboxamide group —CONH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is3; and n is 2. In one embodiment, the compound is general formula II,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a caboxamide group—CONH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 2. In oneembodiment, the compound is general formula II, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 is sulfo;each of R11 and R12 is a caboxamide group —CONH—P where P is a PEGgroup; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a caboxamide group —CONH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is3; and n is 2. In one embodiment, the compound is general formula II,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a caboxamide group—CONH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 2.

In one embodiment, the compound is general formula II, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 issulfo; each of R11 and R12 is a caboxamide group —CONH—P where P is aPEG group; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a caboxamide group —CONH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is3; and n is 3. In one embodiment, the compound is general formula II,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a caboxamide group—CONH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 3. In oneembodiment, the compound is general formula II, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 is sulfo;each of R11 and R12 is a caboxamide group —CONH—P where P is a PEGgroup; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a caboxamide group —CONH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is3; and n is 3. In one embodiment, the compound is general formula II,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a caboxamide group—CONH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 3.

In one embodiment, the compound is

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, heteroaliphatic with terminal SO₃, aPEG group P—Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P—Z, and a caboxamide group-L-CONH—P—Z, and Z is selected from H, a CH₃ group, an alkyl group, or aheteroalkyl group; each of R⁷, R⁸, R¹³, and R¹⁴ is the same or differentand is independently selected from the group consisting of H, SO₃, a PEGgroup P—Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group —SO₂NH—P—Z, and a caboxamide group—CONH—P—Z, and Z is selected from H, a CH₃ group, an alkyl group, aheteroalkyl group, or —CO—NHS; X is selected from the group consistingof —OH, —SH, —NH₂, —NH—NH₂, —F, —Cl, —Br, I, —NHShydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O-STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, —NR-L-NH—CO—CH2-I,imidazole, azide, —NR-L-O—NH2, and —NR-L-O—CO—NHS, where R is —H or analiphatic or heteroaliphatic group, and L is selected from the groupconsisting of a divalent linear (—(CH₂)_(t)—, t=0 to 15), crossed, orcyclic alkyl group optionally substituted by at least one oxygen atomand/or sulfur atom; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or othercation(s) needed to compensate the negative charge brought by thecyanine; m is an integer from 0 to 5 inclusive; p is an integer from 1to 6 inclusive; and n is an integer from 1 to 3 inclusive.

In one embodiment, the compound is general formulas IIIa or IIIb,collectively termed general formula III where each of R1, R5, and R6 ismethyl; R2 is a PEG group; each of R7, R8, R11, and R12 is H; each ofR13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is0; p is 1; and n is 1. In one embodiment, the compound is generalformula III where each of R1, R5, and R6 is methyl; R2 is a PEG group;each of R7, R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is—OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is 2; and n is 1. Inone embodiment, the compound is general formula III, where each of R1,R5, and R6 is methyl; R2 is a PEG group; each of R7, R8, R11, and R12 isH; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 2; p is 3; and n is 1. In one embodiment, thecompound is general formula III, where each of R1, R5, and R6 is methyl;R2 is a PEG group; each of R7, R8, R11, and R12 is H; each of R13 andR14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; p is4; and n is 1. In one embodiment, the compound is general formula III,where each of R1, R5, and R6 is methyl; R2 is a PEG group; each of R7,R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 4; p is 5; and n is 1. In oneembodiment, the compound is general formula III, where each of R1, R5,and R6 is methyl; R2 is a PEG group; each of R7, R8, R11, and R12 is H;each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 5; p is 6; and n is 1.

In one embodiment, the compound is general formula III, where each ofR1, R5, and R6 is methyl; R2 is a PEG group; each of R7, R8, R11, andR12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 2. In one embodiment, thecompound is general formula III, where each of R1, R5, and R6 is methyl;R2 is a PEG group; each of R7, R8, R11, and R12 is H; each of R13 andR14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is2; and n is 2. In one embodiment, the compound is general formula III,where each of R1, R5, and R6 is methyl; R2 is a PEG group; each of R7,R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 2. In oneembodiment, the compound is general formula III, where each of R1, R5,and R6 is methyl; R2 is a PEG group; each of R7, R8, R11, and R12 is H;each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 2. In one embodiment, thecompound is general formula III, where each of R1, R5, and R6 is methyl;R2 is a PEG group; each of R7, R8, R11, and R12 is H; each of R13 andR14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; p is5; and n is 2. In one embodiment, the compound is general formula III,where each of R1, R5, and R6 is methyl; R2 is a PEG group; each of R7,R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 2.

In one embodiment, the compound is general formula III, where each ofR1, R5, and R6 is methyl; R2 is a PEG group; each of R7, R8, R11, andR12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 3. In one embodiment, thecompound is general formula III, where each of R1, R5, and R6 is methyl;R2 is a PEG group; each of R7, R8, R11, and R12 is H; each of R13 andR14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is2; and n is 3. In one embodiment, the compound is general formula III,where each of R1, R5, and R6 is methyl; R2 is a PEG group; each of R7,R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 3. In oneembodiment, the compound is general formula III, where each of R1, R5,and R6 is methyl; R2 is a PEG group; each of R7, R8, R11, and R12 is H;each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 3. In one embodiment, thecompound is general formula III, where each of R1, R5, and R6 is methyl;R2 is a PEG group; each of R7, R8, R11, and R12 is H; each of R13 andR14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; p is5; and n is 3. In one embodiment, the compound is general formula III,where each of R1, R5, and R6 is methyl; R2 is a PEG group; each of R7,R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 3.

In one embodiment, the compound is general formula III, where each ofR1, R5, and R6 is methyl; R2 is a PEG group; each of R7, R8, R11, andR12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 1. In one embodiment, thecompound is general formula III, where each of R1, R5, and R6 is methyl;R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is2; and n is 1. In one embodiment, the compound is general formula III,where each of R1, R5, and R6 is methyl; R2 is a PEG group; each of R7,R8, R11, and R12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 1. In oneembodiment, the compound is general formula III, where each of R1, R5,and R6 is methyl; R2 is a PEG group; each of R7, R8, R11, and R12 issulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 1. In one embodiment, thecompound is general formula III, where each of R1, R5, and R6 is methyl;R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; p is5; and n is 1. In one embodiment, the compound is general formula III,where each of R1, R5, and R6 is methyl; R2 is a PEG group; each of R7,R8, R11, and R12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 1.

In one embodiment, the compound is general formula III, where each ofR1, R5, and R6 is methyl; R2 is a PEG group; each of R7, R8, R11, andR12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 2. In one embodiment, thecompound is general formula III, where each of R1, R5, and R6 is methyl;R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is2; and n is 2. In one embodiment, the compound is general formula III,where each of R1, R5, and R6 is methyl; R2 is a PEG group; each of R7,R8, R11, and R12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 2. In oneembodiment, the compound is general formula III, where each of R1, R5,and R6 is methyl; R2 is a PEG group; each of R7, R8, R11, and R12 issulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 2. In one embodiment, thecompound is general formula III, where each of R1, R5, and R6 is methyl;R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; p is5; and n is 2. In one embodiment, the compound is general formula III,where each of R1, R5, and R6 is methyl; R2 is a PEG group; each of R7,R8, R11, and R12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 2.

In one embodiment, the compound is general formula III, where each ofR1, R5, and R6 is methyl; R2 is a PEG group; each of R7, R8, R11, andR12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 3. In one embodiment, thecompound is general formula III, where each of R1, R5, and R6 is methyl;R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is2; and n is 3. In one embodiment, the compound is general formula III,where each of R1, R5, and R6 is methyl; R2 is a PEG group; each of R7,R8, R11, and R12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 3. In oneembodiment, the compound is general formula III, where each of R1, R5,and R6 is methyl; R2 is a PEG group; each of R7, R8, R11, and R12 issulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 3. In one embodiment, thecompound is general formula III, where each of R1, R5, and R6 is methyl;R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; p is5; and n is 3. In one embodiment, the compound is general formula III,where each of R1, R5, and R6 is methyl; R2 is a PEG group; each of R7,R8, R11, and R12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 3.

In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 1. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; eachof R13 and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1;p is 2; and n is 1. In one embodiment, the compound is general formulaIII, where each of R5 and R6 is methyl; each of R1 and R2 is a PEGgroup; each of R7, R8, R11, and R12 is sulfo; each of R13 and R14 is H;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 1.In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 1. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; eachof R13 and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4;p is 5; and n is 1. In one embodiment, the compound is general formulaIII, where each of R5 and R6 is methyl; each of R1 and R2 is a PEGgroup; each of R7, R8, R11, and R12 is sulfo; each of R13 and R14 is H;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 1.

In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 2. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; eachof R13 and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1;p is 2; and n is 2. In one embodiment, the compound is general formulaIII, where each of R5 and R6 is methyl; each of R1 and R2 is a PEGgroup; each of R7, R8, R11, and R12 is sulfo; each of R13 and R14 is H;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 2.In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 2. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; eachof R13 and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4;p is 5; and n is 2. In one embodiment, the compound is general formulaIII, where each of R5 and R6 is methyl; each of R1 and R2 is a PEGgroup; each of R7, R8, R11, and R12 is sulfo; each of R13 and R14 is H;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 2.

In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is sulfo; each of R13 and R14 is H; X is —OH, NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 3. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; eachof R13 and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1;p is 2; and n is 3. In one embodiment, the compound is general formulaIII, where each of R5 and R6 is methyl; each of R1 and R2 is a PEGgroup; each of R7, R8, R11, and R12 is sulfo; each of R13 and R14 is H;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 3.In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is sulfo; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 3. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is sulfo; eachof R13 and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4;p is 5; and n is 3. In one embodiment, the compound is general formulaIII, where each of R5 and R6 is methyl; each of R1 and R2 is a PEGgroup; each of R7, R8, R11, and R12 is sulfo; each of R13 and R14 is H;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 3.

In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 1. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is H; each ofR13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is1; p is 2; and n is 1. In one embodiment, the compound is generalformula III, where each of R5 and R6 is methyl; each of R1 and R2 is aPEG group; each of R7, R8, R11, and R12 is H; each of R13 and R14 issulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; p is 3; and nis 1. In one embodiment, the compound is general formula III, where eachof R5 and R6 is methyl; each of R1 and R2 is a PEG group; each of R7,R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; p is 4; and n is 1. In oneembodiment, the compound is general formula III, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11, andR12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; p is 5; and n is 1. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is H; each ofR13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is5; p is 6; and n is 1.

In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 2. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is H; each ofR13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is1; p is 2; and n is 2. In one embodiment, the compound is generalformula III, where each of R5 and R6 is methyl; each of R1 and R2 is aPEG group; each of R7, R8, R11, and R12 is H; each of R13 and R14 issulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; p is 3; and nis 2. In one embodiment, the compound is general formula III, where eachof R5 and R6 is methyl; each of R1 and R2 is a PEG group; each of R7,R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; p is 4; and n is 2. In oneembodiment, the compound is general formula III, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11, andR12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; p is 5; and n is 2.

In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 5; p is 6; and n is 2.

In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11,and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 3. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is H; each ofR13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is1; p is 2; and n is 3. In one embodiment, the compound is generalformula III, where each of R5 and R6 is methyl; each of R1 and R2 is aPEG group; each of R7, R8, R11, and R12 is H; each of R13 and R14 issulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; p is 3; and nis 3. In one embodiment, the compound is general formula III, where eachof R5 and R6 is methyl; each of R1 and R2 is a PEG group; each of R7,R8, R11, and R12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; p is 4; and n is 3. In oneembodiment, the compound is general formula III, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7, R8, R11, andR12 is H; each of R13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; p is 5; and n is 3. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7, R8, R11, and R12 is H; each ofR13 and R14 is sulfo; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is5; p is 6; and n is 3.

In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 issulfo; each of R11 and R12 is a sulfonamide group —SO₂NH—P where P is aPEG group; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 1. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a sulfonamide group —SO₂NH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is2; and n is 1. In one embodiment, the compound is general formula III,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a sulfonamide group—SO₂NH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 1. In oneembodiment, the compound is general formula III, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 is sulfo;each of R11 and R12 is a sulfonamide group —SO₂NH—P where P is a PEGgroup; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 1. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a sulfonamide group —SO₂NH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; p is5; and n is 1. In one embodiment, the compound is general formula III,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a sulfonamide group—SO₂NH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 1.

In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 issulfo; each of R11 and R12 is a sulfonamide group —SO₂NH—P where P is aPEG group; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 2. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a sulfonamide group —SO₂NH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is2; and n is 2. In one embodiment, the compound is general formula III,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a sulfonamide group—SO₂NH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 2. In oneembodiment, the compound is general formula III, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 is sulfo;each of R11 and R12 is a sulfonamide group —SO₂NH—P where P is a PEGgroup; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 2. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a sulfonamide group —SO₂NH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; p is5; and n is 2. In one embodiment, the compound is general formula III,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a sulfonamide group—SO₂NH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 2.

In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 issulfo; each of R11 and R12 is a sulfonamide group —SO₂NH—P where P is aPEG group; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 3. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a sulfonamide group —SO₂NH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is2; and n is 3. In one embodiment, the compound is general formula III,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a sulfonamide group—SO₂NH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 3. In oneembodiment, the compound is general formula III, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 is sulfo;each of R11 and R12 is a sulfonamide group —SO₂NH—P where P is a PEGgroup; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 3. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a sulfonamide group —SO₂NH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; p is5; and n is 3. In one embodiment, the compound is general formula III,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a sulfonamide group—SO₂NH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 3.

In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 issulfo; each of R11 and R12 is a caboxamide group —CONH—P where P is aPEG group; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 1. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a caboxamide group —CONH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is2; and n is 1. In one embodiment, the compound is general formula III,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a caboxamide group—CONH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 1. In oneembodiment, the compound is general formula III, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 is sulfo;each of R11 and R12 is a caboxamide group —CONH—P where P is a PEGgroup; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 1. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a caboxamide group —CONH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; p is5; and n is 1. In one embodiment, the compound is general formula whereeach of R5 and R6 is methyl; each of R1 and R2 is a PEG group; each ofR7 and R8 is sulfo; each of R11 and R12 is a caboxamide group —CONH—Pwhere P is a PEG group; each of R13 and R14 is H; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 1.

In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 issulfo; each of R11 and R12 is a caboxamide group —CONH—P where P is aPEG group; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 2. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a caboxamide group —CONH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is2; and n is 2. In one embodiment, the compound is general formula III,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a caboxamide group—CONH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 2. In oneembodiment, the compound is general formula III, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 is sulfo;each of R11 and R12 is a caboxamide group —CONH—P where P is a PEGgroup; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 2. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a caboxamide group —CONH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; p is5; and n is 2. In one embodiment, the compound is general formula III,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a caboxamide group—CONH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 2.

In one embodiment, the compound is general formula III, where each of R5and R6 is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 issulfo; each of R11 and R12 is a caboxamide group —CONH—P where P is aPEG group; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 3. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a caboxamide group —CONH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is2; and n is 3. In one embodiment, the compound is general formula III,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a caboxamide group—CONH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 3. In oneembodiment, the compound is general formula III, where each of R5 and R6is methyl; each of R1 and R2 is a PEG group; each of R7 and R8 is sulfo;each of R11 and R12 is a caboxamide group —CONH—P where P is a PEGgroup; each of R13 and R14 is H; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 3. In one embodiment, thecompound is general formula III, where each of R5 and R6 is methyl; eachof R1 and R2 is a PEG group; each of R7 and R8 is sulfo; each of R11 andR12 is a caboxamide group —CONH—P where P is a PEG group; each of R13and R14 is H; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; p is5; and n is 3. In one embodiment, the compound is general formula III,where each of R5 and R6 is methyl; each of R1 and R2 is a PEG group;each of R7 and R8 is sulfo; each of R11 and R12 is a caboxamide group—CONH—P where P is a PEG group; each of R13 and R14 is H; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 3.

In one embodiment, the compound is 579 Compound 1

579 Compound 1(6-((E)-2-((E)-3-(3-(2-methoxyethyl)-1-methyl-6,8-disulfonato-1-(3-sulfonatopropyl)-1H-benzo[e]indol-3-ium-2-yl)allylidene)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains an ethylene glycol on the indole N of the left heterocycle,i.e., a methylated ethylene glycol. The methyl group on the ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups.

In embodiments, e.g., for functional assays, the inventive compounds areactivated. Activation of the compound adds a chemical moiety such thatthe compound is in a form that can be conjugated to a biological moiety.Examples of chemical moieties for activation are described below withreference to activation of 579 Compound 1, but one skilled in the artappreciates that activation is not limited to these examples. Onenon-limiting example of an activated compound is the NHS-ester of 579Compound 1, shown below:

One non-limiting example of a NHS-ester of 579 Compound 1, according togeneral formula III, where m=1 and p=1, is shown below:

One non-limiting example of a NHS-ester of 579 Compound 1, according togeneral formula III, where m=1 and p=2, is shown below:

One non-limiting example of a NHS-ester of 579 Compound 1, according togeneral formula III, where m=1 and p=3, is shown below:

One non-limiting example of a NHS-ester of 579 Compound 1, according togeneral formula III, where m=1 and p=4, is shown below:

One non-limiting example of a NHS-ester of 579 Compound 1, according togeneral formula III, where m=1 and p=5, is shown below:

One non-limiting example of a NHS-ester of 579 Compound 1, according togeneral formula III, where m=1 and p=6, is shown below:

One non-limiting example of an activated 579 Compound 1 is atetrafluorophenyl (TFP)-ester form of 579 Compound 1, shown below:

One non-limiting example of an activated 579 Compound 1 is asulfotetrafluorophenyl (STP)-ester form of 579 Compound 1, shown below:

One non-limiting example of an activated 579 Compound 1 is a hydrazideform of 579 Compound 1,

One non-limiting example of an activated 579 Compound 1 is a maleimideform of 579 Compound 1, shown below:

In one embodiment, the compound is 579 Compound 2

579 Compound 2(6-((E)-2-((E)-3-(3-(2-(2-methoxyethoxy)ethyl)-1-methyl-6,8-disulfonato-1-(3-sulfonatopropyl)-1H-benzo[e]indol-3-ium-2-yl)allylidene)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 579 Compound 2 is activated as described above.

In one embodiment, the compound is 579 Compound 3

579 Compound 3(6-((E)-2-((E)-3-(3-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-1-methyl-6,8-disulfonato-1-(3-sulfonatopropyl)-1H-benzo[e]indol-3-ium-2-yl)allylidene)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 579 Compound 3 is activated as described above.

In one embodiment, the compound is 579 Compound 4

579 Compound 4(6-((E)-1,1-dimethyl-2-((E)-3-(1-methyl-6,8-disulfonato-1-(3-sulfonatopropyl)-3-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indol-3-ium-2-yl)allylidene)-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 579 Compound 4 is activated as described above.

In one embodiment, the compound is 579 Compound 5

579 Compound 5(6-((E)-2-((E)-3-(3-(2,5,8,11,14-pentaoxahexadecan-16-yl)-1-methyl-6,8-disulfonato-1-(3-sulfonatopropyl)-1H-benzo[e]indol-3-ium-2-yl)allylidene)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 579 Compound 5 is activated as described above.

In one embodiment, the compound is 579 Compound 6

579 Compound 6(6-((E)-1,1-dimethyl-2-((E)-3-(1-methyl-3-(2,5,8,11,14,17-hexaoxanonadecan-19-yl)-6,8-disulfonato-1-(3-sulfonatopropyl)-1H-benzo[e]indol-3-ium-2-yl)allylidene)-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 579 Compound 6 is activated as described above.

In embodiments, the degree and/or location of sulfonation is varied to,e.g., vary the compound's degree of hydrophilicity or hydrophobicity.One non-limiting example is a monosulfonate form of 579 Compound 1,shown below:

One non-limiting example is a disulfonate form of 579 Compound 1, shownbelow:

One non-limiting example is a trisulfonate form of 579 Compound 1, shownbelow:

One non-limiting example is a tetrasulfonate form of 579 Compound 1,shown below:

One non-limiting example is a pentasulfonate form of 579 Compound 1,shown below:

In one embodiment, the compound is 579 Compound 1/2 (PEG₄)

One non-limiting example of 579 Compound 1/2 (PEG₄)(2-((1E,3E)-3-(3-(5-carboxypentyl)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-2(3H)-ylidene)prop-1-enyl)-3-(2-methoxyethyl)-1-methyl-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indolium-6,8-disulfonate)contains an ethylene glycol on the indole N of the left heterocycle,i.e., a methylated ethylene glycol, and a methylated PEG₄ group. Themethyl group on the ethylene glycol/PEG prevents the terminal —OH fromoxidation. Oxidation is known to occur, over time, on an unprotected PEGterminus. Adding a methyl ether provides this protection, and preventsreaction with electrophilic reactive groups.

In embodiments, e.g., for functional assays, the inventive compounds areactivated. Activation of the compound adds a chemical moiety such thatthe compound is in a form that can be conjugated to a biological moiety.Examples of chemical moieties for activation are described below withreference to activation of 579 Compound 1/2 (PEG₄), but one skilled inthe art appreciates that activation is not limited to these examples.One non-limiting example of an activated compound is the NHS-ester of579 Compound 1/2 (PEG₄), shown below:

One non-limiting example of a NHS-ester of 579 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=1, is shown below:

One non-limiting example of a NHS-ester of 579 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=2, is shown below:

One non-limiting example of a NHS-ester of 579 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=3, is shown below:

One non-limiting example of a NHS-ester of 579 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=4, is shown below:

One non-limiting example of a NHS-ester of 579 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=5, is shown below:

One non-limiting example of a NHS-ester of 579 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=6, is shown below:

One non-limiting example of an activated 579 Compound 1/2 (PEG₄) is atetrafluorophenyl (TFP)-ester form of 579 Compound 1, shown below:

One non-limiting example of an activated 579 Compound 1/2 (PEG₄) is asulfotetrafluorophenyl (STP)-ester form of 579 Compound 1, shown below:

One non-limiting example of an activated 579 Compound 1/2 is a hydrazideform of 579 Compound 1 (PEG₄), shown below:

One non-limiting example of an activated 579 Compound 1/2 (PEG₄) is amaleimide form of 579 Compound 1, shown below:

In one embodiment, the compound is 579 Compound 2/2 (PEG₄)

One non-limiting example of 579 Compound 2/2 (PEG₄)(2-((1E,3E)-3-(3-(5-carboxypentyl)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-2(3H)-ylidene)prop-1-enyl)-3-(2-(2-methoxyethoxy)ethyl)-1-methyl-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indolium-6,8-disulfonate)contains a (poly)ethylene glycol on the indole N of the left heterocycleand a PEG₄ group on the indole C. The methyl group on the (poly)ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups. For functional assays, 579 Compound 2/2 (PEG₄) isactivated as described above.

In one embodiment, the compound is 579 Compound 3/2 (PEG₄)

One non-limiting example of 579 Compound 3/2 (PEG₄)(2-((1E,3E)-3-(3-(5-carboxypentyl)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-2(3H)-ylidene)prop-1-enyl)-3-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-1-methyl-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indolium-6,8-disulfonate)contains a (poly)ethylene glycol on the indole N of the left heterocycleand a PEG₄ group on the indole C. The methyl group on the ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups. For functional assays, 579 Compound 3/2 (PEG₄) isactivated as described above.

In one embodiment, the compound is 579 Compound 4/2 (PEG₄)

One non-limiting example of 579 Compound 4/2 (PEG₄)(2-((1E,3E)-3-(3-(5-carboxypentyl)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-2(3H)-ylidene)prop-1-enyl)-1-methyl-1,3-di(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indolium-6,8-disulfonate)contains a (poly)ethylene glycol on the indole N of the left heterocycleand a PEG₄ group on the indole C. The methyl group on the ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups. For functional assays, 579 Compound 4/2 (PEG₄) isactivated as described above.

In one embodiment, the compound is 579 Compound 5/2 (PEG₄)

One non-limiting example of 579 Compound 5/2 (PEG₄)(2-((1E,3E)-3-(3-(5-carboxypentyl)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-2(3H)-ylidene)prop-1-enyl)-3-(2,5,8,11,14-pentaoxahexadecan-16-yl)-1-methyl-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indolium-6,8-disulfonate)contains a (poly)ethylene glycol on the indole N of the left heterocycleand a PEG₄ group on the indole C. The methyl group on the ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups. For functional assays, 579 Compound 5/2 (PEG₄) isactivated as described above.

In one embodiment, the compound is 579 Compound 6/2 (PEG₄)

One non-limiting example of 579 Compound 6/2 (PEG₄)(2-((1E,3E)-3-(3-(5-carboxypentyl)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-2(3H)-ylidene)prop-1-enyl)-1-methyl-3-(2,5,8,11,14,17-hexaoxanonadecan-19-yl)-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indolium-6,8-disulfonate)contains a (poly)ethylene glycol on the indole N of the left heterocycleand a PEG₄ group on the indole C. The methyl group on the ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups. For functional assays, 579 Compound 6/2 (PEG₄) isactivated as described above.

In embodiments, the degree and/or location of sulfonation is varied to,e.g., vary the compound's degree of hydrophilicity or hydrophobicity.One non-limiting example is a monosulfonate form of 579 Compound 1/2(PEG₄), shown below:

One non-limiting example is a disulfonate form of 579 Compound 1/2(PEG₄), shown below:

One non-limiting example is a trisulfonate form of 579 Compound 1/2(PEG₄), shown below:

One non-limiting example is a tetrasulfonate form of 579 Compound 1/2(PEG₄), shown below:

One non-limiting example is a pentasulfonate form of 579 Compound 1/2(PEG₄), shown below:

In embodiments, the compound contains one or more substitutions of thepolymethine linker. In one embodiment, the compound has

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, heteroaliphatic with terminal SO₃, aPEG group P—Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P—Z, and a caboxamide group-L-CONH—P—Z, and Z is selected from H, CH₃, a CH₃ group, an alkyl group,a heteroalkyl group, or —CO—NHS; each of R₇, R₈, R₁₁, R¹², R¹³, and R¹⁴is the same or different and is independently selected from either H,SO₃, a PEG group P—Z where P is selected from an ethylene glycol group,a diethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P—Z, or a caboxamide group-L-CONH—P—Z, and Z is selected from H, a CH₃ group, an alkyl group, or aheteroalkyl group; X is selected from the group consisting of —OH, —SH,—NH₂, —NH—NH₂, —F, —Cl, —Br, I, —NHS(hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O-STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, —NR-L-NH—CO—CH2-I,imidazole, azide, —NR-L-O—NH2, and —NR-L-O—CO—NHS, where R is —H or analiphatic or heteroaliphatic group, and L is selected from the groupconsisting of a divalent linear (—(CH₂)_(t)—, t=0 to 15), crossed, orcyclic alkyl group optionally substituted by at least one oxygen atomand/or sulfur atom; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or othercation(s) needed to compensate the negative charge brought by thecyanine; m is an integer from 0 to 5 inclusive; o is an integer from 0to 12 inclusive; p is an integer from 1 to 6 inclusive; each of R3 andR4 is the same or different and is independently hydrogen, an aliphaticgroup, a heteroaliphatic group, or a PEG group P—Z where P is selectedfrom an ethylene glycol group, a diethylene glycol group, and apolyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, and Z isselected from H, a CH₃ group, an alkyl group, or a heteroalkyl group; orR3 and R4 together form a cyclic structure where R3 and R4 are joinedusing a divalent structural element selected from the group consistingof —(CH₂)_(q)—, —(CH₂)_(q)O(CH₂)_(q′)—, —(CH₂)_(q)S(CH₂)_(q′),—(CH₂)_(q)CH═CH—, and —OCH═CH— where each of q and q′ is the same ordifferent and is a integer from 2 to 6 inclusive; and Y is selected fromthe group consisting of hydrogen, alkyl, sulfoalkyl, fluorine, chlorine,bromine, a substituted or unsubstituted aryl-, phenoxy-, phenylmercaptofunction, and a PEG group P—Z where P is selected from an ethyleneglycol group, a diethylene glycol group, and a polyethylene glycolgroup, where the polyethylene glycol group is (CH₂CH₂O)_(s), where s isan integer from 3-6 inclusive, and Z is selected from H, CH₃, a CH₃group, an alkyl group, or a heteroalkyl group.

In one embodiment, the compound of general formula IV wherein each of R3and R4 is the same or different and is independently hydrogen, analiphatic group, or a heteroaliphatic group, or R3 and R4 together forma cyclic structure where R3 and R4 are directly joined or joined using adivalent structural element selected from the group consisting of—(CH₂)_(q)— and CH═CH, where q is an integer from 1 to 2 inclusive, toresult in a 3-, 4-, or 5-membered ring.

In various embodiments, an ethylene glycol group, diethylene glycolgroup, and/or a polyethylene glycol group, which will collectively bereferred to as a PEG group, unless specifically defined, may be presentat position(s) in addition to such groups being present on the N atom(s)of the indole structure.

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R1 is anethylene glycol group (PEG₁) terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R2 is anethylene glycol group (PEG₁) terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R8 is anethylene glycol group (PEG₁) terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R8 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R8 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R7 is anethylene glycol group (PEG₁) terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R7 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R7 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R12 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R12 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R12 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R11 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R11 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R11 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R14 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R14 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R14 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R13 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R13 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 579 Compound 1/2 according to general formula II where R13 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

In one embodiment, the compound is 679 Compound 1

679 Compound 1(6-((E)-2-((2E,4E)-5-(3-(2-methoxyethyl)-1-methyl-6,8-disulfonato-1-(3-sulfonatopropyl)-1H-benzo[e]indol-3-ium-2-yl)penta-2,4-dien-1-ylidene)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains an ethylene glycol on the indole N of the left heterocycle,i.e., a methylated ethylene glycol. The methyl group on the ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups.

In embodiments, e.g., for functional assays, the inventive compounds areactivated. Activation of the compound adds a chemical moiety such thatthe compound is in a form that can be conjugated to a biological moiety.Examples of chemical moieties for activation are described below withreference to activation of 679 Compound 1, but one skilled in the artappreciates that activation is not limited to these examples. Onenon-limiting example of an activated compound is the NHS-ester of 679Compound 1, shown below:

One non-limiting example of a NHS-ester of 679 Compound 1, according togeneral formula III, where m=1 and p=1, is shown below:

One non-limiting example of a NHS-ester of 679 Compound 1, according togeneral formula III, where m=1 and p=2, is shown below:

One non-limiting example of a NHS-ester of 679 Compound 1, according togeneral formula III, where m=1 and p=3, is shown below:

One non-limiting example of a NHS-ester of 679 Compound 1, according togeneral formula III, where m=1 and p=4, is shown below:

One non-limiting example of a NHS-ester of 679 Compound 1, according togeneral formula III, where m=1 and p=5, is shown below:

One non-limiting example of a NHS-ester of 679 Compound 1, according togeneral formula III, where m=1 and p=6, is shown below:

One non-limiting example of an activated 679 Compound 1 is atetrafluorophenyl (TFP)-ester form of 679 Compound 1, shown below:

One non-limiting example of an activated 679 Compound 1 is asulfotetrafluorophenyl (STP)-ester form of 679 Compound 1, shown below:

One non-limiting example of an activated 679 Compound 1 is a hydrazideform of 679 Compound 1, shown below:

One non-limiting example of an activated 679 Compound 1 is a maleimideform of 679 Compound 1, shown below:

In one embodiment, the compound is 679 Compound 2

679 Compound 2(6-((E)-2-((2E,4E)-5-(3-(2-(2-methoxyethoxy)ethyl)-1-methyl-6,8-disulfonato-1-(3-sulfonatopropyl)-1H-benzo[e]indol-3-ium-2-yl)penta-2,4-dien-1-ylidene)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains a diethylene glycol on the indole N of the left heterocycle.The methyl group on the ethylene glycol prevents the terminal —OH fromoxidation. Oxidation is known to occur, over time, on an unprotected PEGterminus. Adding a methyl ether provides this protection, and preventsreaction with electrophilic reactive groups. For functional assays, 679Compound 2 is activated as described above.

In one embodiment, the compound is 679 Compound 3

679 Compound 3(6-((E)-2-((2E,4E)-5-(3-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-1-methyl-6,8-disulfonato-1-(3-sulfonatopropyl)-1H-benzo[e]indol-3-ium-2-yl)penta-2,4-dien-1-ylidene)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 679 Compound 3 is activated as described above.

In one embodiment, the compound is 679 Compound 4

679 Compound 4(6-((E)-1,1-dimethyl-2-((2E,4E)-5-(1-methyl-6,8-disulfonato-1-(3-sulfonatopropyl)-3-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indol-3-ium-2-yl)penta-2,4-dien-1-ylidene)-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 679 Compound 4 is activated as described above.

In one embodiment, the compound is 679 Compound 5

679 Compound 5(6-((E)-2-((2E,4E)-5-(3-(2,5,8,11,14-pentaoxahexadecan-16-yl)-1-methyl-6,8-disulfonato-1-(3-sulfonatopropyl)-1H-benzo[e]indol-3-ium-2-yl)penta-2,4-dien-1-ylidene)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 679 Compound 5 is activated as described above.

In one embodiment, the compound is 679 Compound 6

679 Compound 6(6-((E)-1,1-dimethyl-2-((2E,4E)-5-(1-methyl-3-(2,5,8,11,14,17-hexaoxanonadecan-19-yl)-6,8-disulfonato-1-(3-sulfonatopropyl)-1H-benzo[e]indol-3-ium-2-yl)penta-2,4-dien-1-ylidene)-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 649 Compound 6 is activated as described above.

In embodiments, the degree and/or location of sulfonation is varied to,e.g., vary the compound's degree of hydrophilicity or hydrophobicity.One non-limiting example is a monosulfonate form of 679 Compound 1,shown below:

One non-limiting example is a disulfonate form of 679 Compound 1, shownbelow:

One non-limiting example is a trisulfonate form of 679 Compound 1, shownbelow:

One non-limiting example is a tetrasulfonate form of 679 Compound 1,shown below:

One non-limiting example is a pentasulfonate form of 679 Compound 1,shown below:

In one embodiment, the compound is 679 Compound 1/2 (PEG₄)

One non-limiting example of 679 Compound 1/2 (PEG₄)(2-((1E,3E,5E)-5-(3-(5-carboxypentyl)-1-methyl-6,8-disulfonato-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dienyl)-3-(2-methoxyethyl)-1-methyl-1-(3-sulfonatopropyl)-1H-benzo[e]indolium-6,8-disulfonate)contains an ethylene glycol on the indole N of the left heterocycle,i.e., a methylated ethylene glycol, and a PEG₄ group on the indole C.The methyl group on the ethylene glycol prevents the terminal —OH fromoxidation. Oxidation is known to occur, over time, on an unprotected PEGterminus. Adding a methyl ether provides this protection, and preventsreaction with electrophilic reactive groups.

In embodiments, e.g., for functional assays, the inventive compounds areactivated. Activation of the compound adds a chemical moiety such thatthe compound is in a form that can be conjugated to a biological moiety.Examples of chemical moieties for activation are described below withreference to activation of 679 Compound 1/2 (PEG₄), but one skilled inthe art appreciates that activation is not limited to these examples.One non-limiting example of an activated compound is the NHS-ester of679 Compound 1/2 (PEG₄), shown below:

One non-limiting example of a NHS-ester of 679 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=1, is shown below:

One non-limiting example of a NHS-ester of 679 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=2, is shown below:

One non-limiting example of a NHS-ester of 679 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=3, is shown below:

One non-limiting example of a NHS-ester of 679 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=4, is shown below:

One non-limiting example of a NHS-ester of 679 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=5, is shown below:

One non-limiting example of a NHS-ester of 679 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=6, is shown below:

One non-limiting example of an activated 679 Compound 1/2 (PEW is atetrafluorophenyl (TFP)-ester form of 679 Compound 1, shown below:

One non-limiting example of an activated 679 Compound 1/2 (PEG₄) is asulfotetrafluorophenyl (STP)-ester form of 679 Compound 1, shown below:

One non-limiting example of an activated 679 Compound 1/2 (PEG₄) is ahydrazide form of 679 Compound 1, shown below:

One non-limiting example of an activated 679 Compound 1/2 (PEG₄) is amaleimide form of 679 Compound 1, shown below:

In one embodiment, the compound is 679 Compound 2/2 (PEG₄)

One non-limiting example of 679 Compound 2/2 (PEG₄)(2-((1E,3E,5E)-5-(3-(5-carboxypentyl)-1-methyl-6,8-disulfonato-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dienyl)-3-(2-(2-methoxyethoxy)ethyl)-1-methyl-1-(3-sulfonatopropyl)-1H-benzo[e]indolium-6,8-disulfonate)contains a diethylene glycol on the indole N of the left heterocycle anda PEG₄ group on the indole C. The methyl group on the ethylene glycolprevents the terminal —OH from oxidation. Oxidation is known to occur,over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups. For functional assays, 679 Compound 2/2 (PEG₄) isactivated as described above.

In one embodiment, the compound is 679 Compound 3/2 (PEG₄)

One non-limiting example of 679 Compound 3/2 (PEG₄)(2-((1E,3E,5E)-5-(3-(5-carboxypentyl)-1-methyl-6,8-disulfonato-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dienyl)-3-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-1-methyl-1-(3-sulfonatopropyl)-1H-benzo[e]indolium-6,8-disulfonate)contains a (poly)ethylene glycol on the indole N of the left heterocycleand a PEG₄ group on the indole C. The methyl group on the ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups. For functional assays, 679 Compound 3/2 (PEG₄) isactivated as described above.

In one embodiment, the compound is 679 Compound 4/2 (PEG₄)

One non-limiting example of 679 Compound 4/2 (PEG₄)(2-((1E,3E,5E)-5-(3-(5-carboxypentyl)-1-methyl-6,8-disulfonato-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dienyl)-1-methyl-1-(3-sulfonatopropyl)-3-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indolium-6,8-disulfonate)contains a (poly)ethylene glycol on the indole N of the left heterocycleand a PEG₄ group on the indole C. The methyl group on the ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups. For functional assays, 679 Compound 4/2 (PEG₄) isactivated as described above.

In one embodiment, the compound is 679 Compound 5/2 (PEG₄)

One non-limiting example of 679 Compound 5/2 (PEG₄)(2-((1E,3E,5E)-5-(3-(5-carboxypentyl)-1-methyl-6,8-disulfonato-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dienyl)-3-(2,5,8,11,14-pentaoxahexadecan-16-yl)-1-methyl-1-(3-sulfonatopropyl)-1H-benzo[e]indolium-6,8-disulfonate)contains a (poly)ethylene glycol on the indole N of the left heterocycleand a PEG₄ group on the indole C. The methyl group on the ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups. For functional assays, 679 Compound 5/2 (PEG₄) isactivated as described above.

In one embodiment, the compound is 679 Compound 6/2 (PEG₄)

One non-limiting example of 679 Compound 6/2 (PEG₄)(2-((1E,3E,5E)-5-(3-(5-carboxypentyl)-1-methyl-6,8-disulfonato-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dienyl)-1-methyl-3-(2,5,8,11,14,17-hexaoxanonadecan-19-yl)-1-(3-sulfonatopropyl)-1H-benzo[e]indolium-6,8-disulfonate)contains a (poly)ethylene glycol on the indole N of the left heterocycleand a PEG₄ group on the indole C. The methyl group on the ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups. For functional assays, 649 Compound 6/2 (PEG₄) isactivated as described above.

In embodiments, the degree and/or location of sulfonation is varied to,e.g., vary the compound's degree of hydrophilicity or hydrophobicity.One non-limiting example is a monosulfonate form of 679 Compound 1/2(PEG₄), shown below:

One non-limiting example is a disulfonate form of 679 Compound 1/2(PEG₄), shown below:

One non-limiting example is a trisulfonate form of 679 Compound 1/2(PEG₄), shown below:

One non-limiting example is a tetrasulfonate form of 679 Compound 1/2(PEG₄), shown below:

One non-limiting example is a pentasulfonate form of 679 Compound 1/2(PEG₄), shown below:

In embodiments, the compound contains one or more substitutions of thepolymethine linker. In one embodiment, the compound has

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, heteroaliphatic with terminal SO₃, aPEG group P—Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P—Z, and a caboxamide group-L-CONH—P—Z, and Z is selected from H, a CH₃ group, an alkyl group, aheteroalkyl group, or —CO—NHS; each of R⁷, R⁸, R¹¹, R¹², R¹³, and R¹⁴ isthe same or different and is independently selected from either H, SO₃,a PEG group P—Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group —SO₂NH—P—Z, or a caboxamide group—CONH—P—Z, and Z is selected from H, a CH₃ group, an alkyl group, aheteroalkyl group, or —CO—NHS; X is selected from the group consistingof —OH, —SH, —NH₂, —NH—NH₂, —F, —Cl, —Br, I, —NHS(hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O-STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, —NR-L-NH—CO—CH2-I,imidazole, azide, —NR-L-O—NH2, and —NR-L-O—CO—NHS, where R is —H or analiphatic or heteroaliphatic group, and L is selected from the groupconsisting of a divalent linear (—(CH₂)_(t)—, t=0 to 15), crossed, orcyclic alkyl group optionally substituted by at least one oxygen atomand/or sulfur atom; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or othercation(s) needed to compensate the negative charge brought by thecyanine; m is an integer from 0 to 5 inclusive; o is an integer from 0to 12 inclusive; p is an integer from 1 to 6 inclusive; each of R3 andR4 is the same or different and is independently hydrogen, an aliphaticgroup, a heteroaliphatic group, a PEG group P—Z where P is selected froman ethylene glycol group, a diethylene glycol group, and a polyethyleneglycol group, where the polyethylene glycol group is (CH₂CH₂O)_(s),where s is an integer from 3-6 inclusive, and Z is selected from H, CH₃,a CH₃ group, an alkyl group, or a heteroalkyl group; or R3 and R4together form a cyclic structure where R3 and R4 are joined using adivalent structural element selected from the group consisting of—(CH₂)_(q)—, —(CH₂)_(q)O(CH₂)_(q), —(CH₂)_(q)S(CH₂)_(q′)—,—(CH₂)_(q)CH═CH—, and —OCH═CH— where each of q and q′ is the same ordifferent and is a integer from 2 to 6 inclusive; and Y is selected fromthe group consisting of hydrogen, alkyl, sulfoalkyl, fluorine, chlorine,bromine, a substituted or unsubstituted aryl-, phenoxy-, phenylmercaptofunction, and a PEG group P—Z where P is selected from an ethyleneglycol group, a diethylene glycol group, and a polyethylene glycolgroup, where the polyethylene glycol group is (CH₂CH₂O)_(s), where s isan integer from 3-6 inclusive, and Z is selected from H, CH₃, a CH₃group, an alkyl group, a heteroalkyl group, or —CO—NHS.

In one embodiment, the compound of general formula V wherein each of R3and R4 is the same or different and is independently hydrogen, analiphatic group, or a heteroaliphatic group, or R3 and R4 together forma cyclic structure where R3 and R4 are directly joined or joined using adivalent structural element selected from the group consisting of—(CH₂)_(q)— and CH═CH, where q is an integer from 1 to 2 inclusive, toresult in a 3-, 4-, or 5-membered ring.

One non-limiting example is a substituted polymethine form of 679Compound 1, shown below:

One non-limiting example is a substituted polymethine form of 679Compound 2, shown below:

One non-limiting example is a substituted polymethine form of 679Compound 3, shown below:

One non-limiting example is a substituted polymethine form of 679Compound 4, shown below:

One non-limiting example is a substituted polymethine form of 679Compound 5, shown below:

One non-limiting example is a substituted polymethine form of 679Compound 6, shown below:

One non-limiting example is a substituted polymethine form of 679Compound 1, shown below:

One non-limiting example is a substituted polymethine form of 679Compound 2, shown below:

One non-limiting example is a substituted polymethine form of 679Compound 3, shown below:

One non-limiting example is a substituted polymethine form of 679Compound 4, shown below:

One non-limiting example is a substituted polymethine form of 679Compound 5, shown below:

One non-limiting example is a substituted polymethine form of 679Compound 6, shown below:

One non-limiting example is a substituted polymethine form of 679Compound 1/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 679Compound 2/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 679Compound 3/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 679Compound 4/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 679Compound 5/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 679Compound 6/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 679Compound 1/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 679Compound 2/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 679Compound 3/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 679Compound 4/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 679Compound 5/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 679Compound 6/2 (PEG₄), shown below:

In various embodiments, an ethylene glycol group, diethylene glycolgroup, and/or a polyethylene glycol group, which will collectively bereferred to as a PEG group, unless specifically defined, may be presentat position(s) in addition to such groups being present on the N atom(s)of the indole structure. One non-limiting example of an additionallyPEG-substituted compound is a 679 Compound 1/2 according to generalformula II where R1 is an ethylene glycol group terminating with amethyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R2 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R8 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R8 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R8 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R7 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R7 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R7 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R12 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R12 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R12 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R11 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R11 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R11 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R13 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R13 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R13 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R14 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R13 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 1/2 according to general formula II where R13 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 4/4 (V08-15173) according to general formula III whereeach of R1 and R2 is a polyethylene glycol (4) group terminating with amethyl group, p=4, X=NHS, and each of R7, R8, R11, and R12 are SO₃,shown below:

One non-limiting example of an additionally PEG-substituted compound isa 679 Compound 4/4 (V10-04152) according to general formula III whereeach of R1 and R2 is a polyethylene glycol (4) group terminating with amethyl group, p=4, X=OH, and each of R13 and R14 are SO₃, shown below:

In one embodiment, the compound is 779 Compound 1

779 Compound 1(6-((E)-2-((2E,4E,6E)-7-(3-(2-methoxyethyl)-1-methyl-6,8-disulfonato-1-(3-sulfonatopropyl)-1H-benzo[e]indol-3-ium-2-yl)hepta-2,4,6-trien-1-ylidene)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains an ethylene glycol on the indole N of the left heterocycle,i.e., a methylated ethylene glycol. The methyl group on the ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups.

In embodiments, e.g., for functional assays, the inventive compounds areactivated. Activation of the compound adds a chemical moiety such thatthe compound is in a form that can be conjugated to a biological moiety.Examples of chemical moieties for activation are described below withreference to activation of 779 Compound 1, but one skilled in the artappreciates that activation is not limited to these examples. Onenon-limiting example of an activated compound is the NHS-ester of 779Compound 1, shown below:

One non-limiting example of a NHS-ester of 779 Compound 1, according togeneral formula III, where m=1 and p=1, is shown below:

One non-limiting example of a NHS-ester of 779 Compound 1, according togeneral formula III, where m=1 and p=2, is shown below:

One non-limiting example of a NHS-ester of 779 Compound 1, according togeneral formula III, where m=1 and p=3, is shown below:

One non-limiting example of a NHS-ester of 779 Compound 1, according togeneral formula III, where m=1 and p=4, is shown below:

One non-limiting example of a NHS-ester of 779 Compound 1, according togeneral formula III, where m=1 and p=5, is shown below:

One non-limiting example of a NHS-ester of 779 Compound 1, according togeneral formula III, where m=1 and p=6, is shown below:

One non-limiting example of an activated 779 Compound 1 is atetrafluorophenyl (TFP)-ester form of 779 Compound 1, shown below:

One non-limiting example of an activated 779 Compound 1 is asulfotetrafluorophenyl (STP)-ester form of 779 Compound 1, shown below:

One non-limiting example of an activated 779 Compound 1 is a hydrazideform of 779 Compound 1, shown below:

One non-limiting example of an activated 779 Compound 1 is a maleimideform of 779 Compound 1, shown below:

In one embodiment, the compound is 779 Compound 2

779 Compound 2(6-((E)-2-((2E,4E,6E)-7-(3-(2-(2-methoxyethoxy)ethyl)-1-methyl-6,8-disulfonato-1-(3-sulfonatopropyl)-1H-benzo[e]indol-3-ium-2-yphepta-2,4,6-trien-1-ylidene)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains a diethylene glycol on the indole N of the left heterocycle.The methyl group on the ethylene glycol prevents the terminal —OH fromoxidation. Oxidation is known to occur, over time, on an unprotected PEGterminus. Adding a methyl ether provides this protection, and preventsreaction with electrophilic reactive groups. For functional assays, 779Compound 2 is activated as described above.

In one embodiment, the compound is 779 Compound 3

779 Compound 3(6-((E)-2-((2E,4E,6E)-7-(3-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-1-methyl-6,8-disulfonato-1-(3-sulfonatopropyl)-1H-benzo[e]indol-3-ium-2-yl)hepta-2,4,6-trien-1-ylidene)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 779 Compound 3 is activated as described above.

In one embodiment, the compound is 779 Compound 4

779 Compound 4(6-((E)-1,1-dimethyl-2-((2E,4E,6E)-7-(1-methyl-6,8-disulfonato-1-(3-sulfonatopropyl)-3-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indol-3-ium-2-yl)hepta-2,4,6-trien-1-ylidene)-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 779 Compound 4 is activated as described above.

In one embodiment, the compound is 779 Compound 5

779 Compound 5(6-((E)-2-((2E,4E,6E)-7-(3-(2,5,8,11,14-pentaoxahexadecan-16-yl)-1-methyl-6,8-disulfonato-1-(3-sulfonatopropyl)-1H-benzo[e]indol-3-ium-2-yl)hepta-2,4,6-trien-1-ylidene)-1,1-dimethyl-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yphexanoate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 779 Compound 5 is activated as described above.

In one embodiment, the compound is 779 Compound 6

779 Compound 6(6-((E)-1,1-dimethyl-2-((2E,4E,6E)-7-(1-methyl-3-(2,5,8,11,14,17-hexaoxanonadecan-19-yl)-6,8-disulfonato-1-(3-sulfonatopropyl)-1H-benzo[e]indol-3-ium-2-yl)hepta-2,4,6-trien-1-ylidene)-6,8-disulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 779 Compound 6 is activated as described above.

In embodiments, the degree and/or location of sulfonation is varied to,e.g., vary the compound's degree of hydroplilicity or hydrophobicity.One non-limiting example is a monosulfonate form of 779 Compound 1,shown below:

One non-limiting example is a disulfonate form of 779 Compound 1, shownbelow:

One non-limiting example is a trisulfonate form of 779 Compound 1, shownbelow:

One non-limiting example is a tetrasulfonate form of 779 Compound 1,shown below:

One non-limiting example is a pentasulfonate form of 779 Compound 1,shown below:

In one embodiment, the compound is 779 Compound 1/2 (PEG₄)

779 Compound 1/2 (PEG₄)(2-((1E,3E,5E,7E)-7-(3-(5-carboxypentyl)-1-methyl-6,8-disulfonato-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indol-2(3H)-ylidene)hepta-1,3,5-trienyl)-3-(2-methoxyethyl)-1-methyl-1-(3-sulfonatopropyl)-1H-benzo[e]indolium-6,8-disulfonate)contains an ethylene glycol on the indole N of the left heterocycle,i.e., a methylated ethylene glycol and a PEG₄ group on the indole C. Themethyl group on the ethylene glycol prevents the terminal —OH fromoxidation. Oxidation is known to occur, over time, on an unprotected PEGterminus. Adding a methyl ether provides this protection, and preventsreaction with electrophilic reactive groups.

In embodiments, e.g., for functional assays, the inventive compounds areactivated. Activation of the compound adds a chemical moiety such thatthe compound is in a form that can be conjugated to a biological moiety.Examples of chemical moieties for activation are described below withreference to activation of 779 Compound 1/2, but one skilled in the artappreciates that activation is not limited to these examples. Onenon-limiting example of an activated compound is the NHS-ester of 779Compound 1/2 (PEG₄), shown below:

One non-limiting example of a NHS-ester of 779 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=1, is shown below:

One non-limiting example of a NHS-ester of 779 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=2, is shown below:

One non-limiting example of a NHS-ester of 779 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=3, is shown below:

One non-limiting example of a NHS-ester of 779 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=4, is shown below:

One non-limiting example of a NHS-ester of 779 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=5, is shown below:

One non-limiting example of a NHS-ester of 779 Compound 1/3 (PEG₄),according to general formula III, where m=1 and p=6, is shown below:

One non-limiting example of an activated 779 Compound 1/2 (PEG₄) is atetrafluorophenyl (TFP)-ester form of 779 Compound 1, shown below:

One non-limiting example of an activated 779 Compound 1/2 (PEG₄) is asulfotetrafluorophenyl (STP)-ester form of 779 Compound 1/2, shownbelow:

One non-limiting example of an activated 779 Compound 1/2 (PEG₄) is ahydrazide form of 779 Compound 1/2, shown below:

One non-limiting example of an activated 779 Compound 1/2 (PEG₄) is amaleimide form of 779 Compound 1/2, shown below:

In one embodiment, the compound is 779 Compound 2/2 (PEG₄)

779 Compound 2/2 (PEG₄)(2-((1E,3E,5E,7E)-7-(3-(5-carboxypentyl)-1-methyl-6,8-disulfonato-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indol-2(3H)-ylidene)hepta-1,3,5-trienyl)-3-(2-(2-methoxyethoxy)ethyl)-1-methyl-1-(3-sulfonatopropyl)-1H-benzo[e]indolium-6,8-disulfonate)contains a diethylene glycol on the indole N of the left heterocycle anda PEG₄ group on the indole C. The methyl group on the (poly)ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups. For functional assays, 779 Compound 2/2 (PEG₄) isactivated as described above.

In one embodiment, the compound is 779 Compound 3/2 (PEG₄)

779 Compound 3/2 (PEG₄)(2-((1E,3E,5E,7E)-7-(3-(5-carboxypentyl)-1-methyl-6,8-disulfonato-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indol-2(3H)-ylidene)hepta-1,3,5-trienyl)-3-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-1-methyl-1-(3-sulfonatopropyl)-1H-benzo[e]indolium-6,8-disulfonate)contains a (poly)ethylene glycol on the indole N of the left heterocycleand a PEG₄ group on the indole C. The methyl group on the (poly)ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups. For functional assays, 779 Compound 3/2 (PEG₄) isactivated as described above.

In one embodiment, the compound is 779 Compound 4/2 (PEG₄)

779 Compound 4/2 (PEG₄)(2-((1E,3E,5E,7E)-7-(3-(5-carboxypentyl)-1-methyl-6,8-disulfonato-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indol-2(3H)-ylidene)hepta-1,3,5-trienyl)-1-methyl-1-(3-sulfonatopropyl)-3-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indolium-6,8-disulfonate)contains a (poly)ethylene glycol on the indole N of the left heterocycleand a PEG₄ group on the indole C. The methyl group on the (poly)ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups. For functional assays, 779 Compound 4/2 (PEG₄) isactivated as described above.

In one embodiment, the compound is 779 Compound 5/2 (PEG₄)

779 Compound 5/2 (PEG₄)(2-((1E,3E,5E,7E)-7-(3-(5-carboxypentyl)-1-methyl-6,8-disulfonato-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indol-2(3H)-ylidene)hepta-1,3,5-trienyl)-3-(2,5,8,11,14-pentaoxahexadecan-16-yl)-1-methyl-1-(3-sulfonatopropyl)-1H-benzo[e]indolium-6,8-disulfonate)contains a (poly)ethylene glycol on the indole N of the left heterocycleand a PEG₄ group on the indole C. The methyl group on the (poly)ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected PEG terminus. Adding a methyl etherprovides this protection, and prevents reaction with electrophilicreactive groups. For functional assays, 779 Compound 5/2 (PEG₄) isactivated as described above.

In one embodiment, the compound is 779 Compound 6/2 (PEG₄)

779 Compound 6/2 (PEG₄)(2-((1E,3E,5E,7E)-7-(3-(5-carboxypentyl)-1-methyl-6,8-disulfonato-1-(2,5,8,11-tetraoxatridecan-13-yl)-1H-benzo[e]indol-2(3H)-ylidene)hepta-1,3,5-trienyl)-1-methyl-3-(2,5,8,11,14,17-hexaoxanonadecan-19-yl)-1-(3-sulfonatopropyl)-1H-benzo[e]indolium-6,8-disulfonate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the (poly)ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 779 Compound 6/2 (PEG₄) is activated as describedabove.

In embodiments, the degree and/or location of sulfonation is varied to,e.g., vary the compound's degree of hydroplilicity or hydrophobicity.One non-limiting example is a monosulfonate form of 779 Compound 1/2(PEG₄), shown below:

One non-limiting example is a disulfonate form of 779 Compound 1/2(PEG₄), shown below:

One non-limiting example is a trisulfonate form of 779 Compound 1/2(PEG₄), shown below:

One non-limiting example is a tetrasulfonate form of 779 Compound 1/2(PEG₄), shown below:

One non-limiting example is a pentasulfonate form of 779 Compound 1/2(PEG₄), shown below:

In embodiments, the compound contains one or more substitutions of thepolymethine linker. In one embodiment, the compound has

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, heteroaliphatic with terminal SO₃, aPEG group P—Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P—Z, and a caboxamide group-L-CONH—P—Z, and Z is selected from H, CH₃, a CH₃ group, an alkyl group,a heteroalkyl group, or —CO—NHS; each of R⁷, R⁸, R¹¹, R¹², R¹³, and R¹⁴is the same or different and is independently selected from either H,SO₃, a PEG group P—Z where P is selected from an ethylene glycol group,a diethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)₅, where s is an integer from 3-6inclusive, a sulfonamide group -L-SO2NH—P—Z, or a caboxamide group-L-CONH—P—Z, and Z is selected from H, a CH₃ group, an alkyl group, aheteroalkyl group, or —CO—NHS; X is selected from the group consistingof —OH, —SH, —NH₂, —NH—NH₂, —F, —Cl, —Br, I, —NHS(hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O-STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, —NR-L-NH—CO—CH2-I,imidazole, azide, —NR-L-O—NH2, and —NR-L-O—CO—NHS, where R is —H or analiphatic or heteroaliphatic group, and L is selected from the groupconsisting of a divalent linear (—(CH₂)_(t)—, t=0 to 15), crossed, orcyclic alkyl group optionally substituted by at least one oxygen atomand/or sulfur atom; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or othercation(s) needed to compensate the negative charge brought by thecyanine; m is an integer from 0 to 5 inclusive; o is an integer from 0to 12 inclusive; p is an integer from 1 to 6 inclusive; each of R3 andR4 is the same or different and is independently hydrogen, an aliphaticgroup, a heteroaliphatic group, or a PEG group P—Z where P is selectedfrom an ethylene glycol group, a diethylene glycol group, and apolyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, and Z isselected from H, CH₃, a CH₃ group, an alkyl group, a heteroalkyl group,or —CO—NHS; or R3 and R4 together form a cyclic structure where R3 andR4 are joined using a divalent structural element selected from thegroup consisting of —(CH₂)_(q)—, —(CH₂)_(q)O(CH₂)_(q′)—,—(CH₂)_(q)S(CH₂)_(q′)—, —(CH₂)_(q)CH═CH—, and —OCH═CH— where each of qand q′ is the same or different and is a integer from 2 to 6 inclusive;and Y is selected from the group consisting of hydrogen, alkyl,sulfoalkyl, fluorine, chlorine, bromine, a substituted or unsubstitutedaryl-, phenoxy-, phenylmercapto function, and a PEG group P—Z where P isselected from an ethylene glycol group, a diethylene glycol group, and apolyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, and Z isselected from H, CH₃, a CH₃ group, an alkyl group, a heteroalkyl group,or —CO—NHS.

In one embodiment, the compound of general formula VI wherein each of R3and R4 is the same or different and is independently hydrogen, analiphatic group, or a heteroaliphatic group, or R3 and R4 together forma cyclic structure where R3 and R4 are directly joined or joined using adivalent structural element selected from the group consisting of—(CH₂)_(q)— and CH═CH, where q is an integer from 1 to 2 inclusive, toresult in a 3-, 4-, or 5-membered ring.

One non-limiting example is a substituted polymethine form of 779Compound 1, shown below:

One non-limiting example is a substituted polymethine form of 779Compound 2, shown below:

One non-limiting example is a substituted polymethine form of 779Compound 3, shown below:

One non-limiting example is a substituted polymethine form of 779Compound 4, shown below:

One non-limiting example is a substituted polymethine form of 779Compound 5, shown below:

One non-limiting example is a substituted polymethine form of 779Compound 6, shown below:

One non-limiting example is a substituted polymethine form of 779Compound 1/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 779Compound 2/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 779Compound 3/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 779Compound 4/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 779Compound 5/2 (PEG₄), shown below:

One non-limiting example is a substituted polymethine form of 779Compound 6/2 (PEG₄), shown below:

In embodiments, an ethylene glycol group, diethylene glycol group,and/or a polyethylene glycol group, collectively referred to as a PEGgroup unless specifically defined, may be present at position(s) inaddition to such groups being present on the N atom(s) of the indolestructure.

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R1 is anethylene glycol group terminating with a methyl group,

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R2 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R8 is anethylene glycol group terminating with a methyl group,

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R8 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R8 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R7 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R7 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R7 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R12 is anethylene glycol group terminating with a methyl group,

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R12 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R12 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R11 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R11 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R11 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R13 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R13 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R13 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R14 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R14 is asulfonamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 779 Compound 1/2 according to general formula II where R14 is acarboxamide group with an ethylene glycol group (PEG₁) terminating witha methyl group, shown below:

The disclosed compounds are useful as chromophores and/or fluorophores.For example, they are used for optical labelling and, therefore, for thequalitative and/or quantitative detection of proteins, nucleic acids,oligomers, DNA, RNA, biological cells, lipids, mono-, oligo- andpolysaccharides, ligands, receptors, polymers, drugs, polymeric beads,etc.

The present compounds, containing the disclosed functionality orfunctionalities, may be synthesized using methods known in the art,e.g., as described as follows with all references expressly incorporatedby reference herein in their entirety.

The core indocyanine structure without additional functionalilities,along with its synthesis, was described by König in U.S. Pat. No.1,524,791 and BP 434875, and included 3-, 5-, and 7-membered polymethinechains.

Synthesis of numerous modifications of the core indocyanine structurehave been described. Such modifications provided variousfunctionalilities, e.g., synthesis of N-isothiocyanato-alkyl- andaromatic-carboxyalkyl-functionalizedindocyanines were described in U.S.Pat. Nos. 5,627,027; 6,048,982; 4,981,977; U.S. Publication No.2006/0199949; Southwick, Anal. Chem. 67 (1995)1742-48).

Synthesis of indocyanines with one or two N-carboxyalkyl functionalitieswere described in U.S. Pat. Nos. 5,268,486; 5,486,616; 5,569,587;5,569,766; and JP 03217837.

Synthesis of indocyanines containing C-carboxyalkyl groups weredescribed in JP 05-313304; U.S. Publication Nos. 2006/0099638,2006/0004188; 2002/0077487; 2002/0064794; U.S. Pat. Nos. 6,977,305 and6,974,873.

Synthesis of indocyanines with N- and C-sulfoalkyl groups were describedin JP 05-313304; WO 2005/044923; U.S. Publication No. 2007/0203343.

Synthesis of indocyanines with mixed C-carboxyalkyl and C-sulfoalkylwere described in EP 1792949 and U.S. Pat. No. 7,745,640.

Synthesis of indocyanaines having a PEG-containing, N-carboxyalkylspacer were described in U.S. Pat. No. 6,939,532.

Functionalization of the N-carboxyalkyl with an amino-functionalizedPEG-alkyl chain, and N- and C-substituted PEG-alkyl chains, weredescribed in U.S. Publication No. 2009/0305410.

Synthesis of various polymethine bridge substitutions, and otherfunctionalizations of indocyanines, were described in Strekowski,Heterocyclic Polymethine Dyes: Synthesis, Properties and Applications,(2008) Springer-Verlag, Berlin Heidelberg; Gragg, “Synthesis ofNear-Infrared Heptamethine Cyanine Dyes” (2010). Chemistry Theses. Paper28; Patonay et al. (2004) Noncovalent Labeling of Biomolecules with Redand Near-Infrared Dyes. Molecules 9 (2004) 40-49; and U.S. Pat. No.7,172,907. Examples 1-5 disclose synthesis reactions for the compounds.

In one embodiment, the compound is synthesized by a condensationreaction, known to one skilled in the art, of the two differentlysubstituted indole heterocycles separated by a (poly)methine linker orbridge, e.g., C1, C3, or C5. Other synthesis methods are possible. Asonly one example, one of the indole heterocycles is first reacted withthe C1, C3, or C5 linker. The 1:1 condensation product is isolated, andthen condensed with the second indole heterocycle to result in thecyanine compound. The sequence of reacting the indole heterocycles isirrelevant. Thus, a plurality of differently functionalized, stronglyhydrophilic, diastereomeric compounds, that differ in total charge andspecificity/reactivity of the active groups used for theirimmobilization, can be easily prepared.

Conjugates of the compounds are prepared by covalently coupling thecompounds to a biomolecule using the functional substituent on theN-position of the indole ring. This functional substituent is activatedby routine protein chemistry reaction methods known to one skilled inthe art. The activated compound may be converted to, without limitation,an N-hydroxysuccinimide (NHS)-ester, an acid fluoride, atetrafluorophenyl (TFP)- or sulfotetrafluorophenyl (STP)-ester, aniodoacetyl group, a maleimide, a hydrazide, a sulfonyl chloride, aphenylazide. Methods for preparing such compounds are known to oneskilled in the art. In one embodiment, the activated substituent is thenreacted with an amino group on the biomolecule under conditions to formthe linkage. In one embodiment, a non-activated carboxyl group on theN-position of the indole in the compound is coupled to an amine using acarbodimide.

In one embodiment, a N-hydroxysuccinimidyl ester (X=—NHS) of a compoundwas formed as follows: 20 μmol dye with X═OH (carboxyalkyl group), 8 mg(40 μmol) dicyclohexylcarbodiimide, and 5 mg (40 μmol)N-hydroxysuccinimide were dissolved in 2 ml of DMF and 100 μl water. Sixμl (40 μmol) triethylamine was then added. The reaction mixture wasstirred at room temperature (about 20° C. to about 22° C.) for 24 hoursand then filtered. The solvent was removed and the residue was washedwith diethylether. The reaction proceeded quantitatively.

In one embodiment, a maleimide (X=—NH—CH₂CH₂-maleimide) of a compoundwas formed as follows: 20 μmol dye with X=—NHS(N-hydroxysuccinimid-ester) was dissolved in 2 ml DMF and 100 water andmixed with 7.6 mg (30 μmol) 2-maleimidoethylamine-trifluoracetate and 5μl (30 μmol) N-ethyldiisopropyl-amine. The reaction mixture was stirredfor three hours at room temperature (about 20° C. to about 22° C.). Thesolvent was evaporated under reduced pressure. The residue was washedwith diethylether and acetone and dried in vacuum. The reactionproceeded quantitatively.

In one embodiment, a iodoacetamide (X=—NH—CH₂CH₂—NH—CO—CH₂—I) of acompound was formed as follows: 20 μmol dye with X=—NHS(N-hydroxysuccinimid-ester) was dissolved in 2 ml DMF and 100 μl water,followed by the addition of 40 mg (300 μmol)ethylendiamindihydrochloride and 26 μl (150 μmol)N-ethyldiisopropyl-amine. The reaction mixture was stirred for threehours at room temperature (about 20° C. to about 22° C.). The solventwas then evaporated under reduced pressure, the residue was dissolved inmethanol, and the ethylendiamindihydrochloride was removed byfiltration. The methanol was evaporated under reduced pressure. Theresidue was dissolved in 2 ml dry DMF, followed by then addition of 7 mg(25 μmol) N-succinimidyl iodoacetate and 4 μl (25 μmol)N-ethyldiisopropyl-amine. The reaction mixture was stirred for threehours at room temperature. The solvent was evaporated under reducedpressure and the residue was purified via reverse phase HPLC.

In one embodiment, a hydroxyl group, such as a terminal hydroxyl group,can be subsequently activated to a reactive derivative able to linkwith, for example, proteins and other molecules. Examples of activatinggroups include tosyl chloride (TsCl), tresyl chloride (TrCl),disuccinimidyl carbonate (DSC), divinyl sulfone, bis-epoxy compounds,carbonyl diimidazole (CDI), 2-fluoro-1-methylpyridinium (FMP), andtrichloro-s-triazine (TsT). In one embodiment, the hydroxyl group isactivated to a succinimidyl carbonate, which is reactive with amines.For example, disuccinimidyl carbonate (DSC) can be used to createamine-reactive groups from hydroxyls in a single step, as described inWilchek, M. and Miron, T. (1985) (Activation of Sepharose withN,N′-disuccinimidyl carbonate. Applied Biochemistry and Biotechnology11, 191-193). DSC reacts with a hydroxyl group, such as at the end of aPEG chain of the described compounds, to directly form a reactive esterwith loss of one molecule of NHS. This reactive group, which is an NHScarbonate, can be used to couple a described compound toamine-containing molecules, such as proteins. For example, a reaction ofan NHS carbonate with an amine creates a carbamate linkage (a urethanebond), which is as stable as the amide bonds formed from the reaction ofan NHS ester with an amine. In one embodiment, a terminal hydroxyl groupof a PEG-containing compound can be activated with DSC to provide an NHScarbonate reactive group for coupling to amine-containing molecules. Inone embodiment, the group X, as described in the disclosed generalformulas, is a spacer arm that terminates in a hydroxyl group, such as aPEG group, which also can be activated with DSC to create the NHScarbonate, i.e., when X=—NR-L-O—CO—NHS.

Coupling between the compound and the biomolecule may be performed asfollows. The compound is reacted with the biomolecule in an organic oraqueous solution at a pH between pH 5 and pH 12, inclusive. The compoundneed not be dissolved in an organic solvent, such as dimethyl formamide(DMF) or dimethyl sulfoxide (DMSO) prior to adding the biomolecule. Inone embodiment, coupling reaction may be performed in a 100% aqueoussolution. In one embodiment, the coupling reaction occurs at roomtemperature (about 20° C. to about 22° C.).

To form a dye composition, at least one biocompatible excipient is addedto the compound, as known to one of ordinary skill in the art.Excipients include but are not limited to buffers, solubility enhancingagents, stabilizing agents, etc.

In one embodiment, a kit for performing an assay method comprises adisclosed compound, and instructions for performing the method using thecompound.

The disclosed activated compounds (i.e., the compound modified with areactive group) are useful to label macromolecules (e.g., antibodies,streptavidin, etc) using methods known to one skilled in the art, e.g.,as disclosed in Hermanson, Bioconjugate Techniques, 2nd Ed., London,Elsevier Inc. 2008. The reaction is carried out for one hour to twohours at room temperature (about 20° C. to about 22° C.), and thendesalted by dialyzing against several changes of phosphate bufferedsaline (pH 7.2) or purified by gel filtration to remove the unreactedfluorescent dye. The resulting compound-biomolecule conjugate is usefulin applications such as detection of specific proteins in immunoassays,sugars in glycoproteins with lectins, protein-protein interactions,oligonucleotides in nucleic acid, hybridization, and in electrophoreticmobility shift assays (EMSA).

The resulting compound-biomolecule conjugates exhibit fluorescentproperties. They may be used in optical, including fluorescence optical,qualitative and quantitative determination methods. Examples of suchmethods include, but are not limited to, microscopy, immunoassays,hybridization methods, chromatographic and electrophoretic methods,fluorescence resonance energy transfer (FRET) systems, high throughputscreenings, analysis of receptor-ligand interactions on a microarray,etc.

Compounds of any of the embodiments can be used as dyes for opticallabelling of organic or inorganic biomolecules, also referred to asrecognition units. Recognition units are molecules having specificityand/or affinity for a specific group of molecules. Examples ofrecognition units include, but are not limited to, antibodies that haveaffinity for antigens, enzymes that bind and/or react with a specificbond or bonds within a sequence of amino acids in a peptide or reactwith a substrate, cofactors such as metals that enhance or inhibitspecific interactions, lectins that bind specific sugars or sugarsequences (e.g., oligosaccharides, polysaccharides, dextrans, etc.),biotin binding proteins such as avidin and streptavidin that bind biotinand biotinylated molecules, antibody binding proteins such as Protein A,Protein G, Protein A/G and Protein L, sequences of amino acids or metalsthat have affinity for each other (e.g., histidine sequences bind nickelor copper, phosphate containing proteins that bind gallium, aluminium,etc.), specific sequences of nucleic acids such as DNA and/or RNAoligonucleotides that have affinity for proteins, specific sequences ofamino acids that have affinity for DNA and/or RNA, haptens, carotenoids,hormones (e.g., neurohormone), neurotransmitters, growth factors,toxins, biological cells, lipids, receptor binding drugs or organic orinorganic polymeric carrier materials, fluorescent proteins such asphycobilliproteins (e.g., phycoethrin, allophycocyanin), etc. The ionicinteractions between these recognition units and the disclosed compoundsresults in labeling of the recognition units. The recognition unit andcompound can be covalently bound. The result is a conjugate forqualitative or quantitative determination of various biomaterials orother organic or inorganic materials using optical methods.

The inventive compounds and/or conjugates are useful in optical,including fluorescence optical, qualitative and/or quantitativedetermination methods to diagnose properties of cells (molecularimaging), in biosensors (point of care measurements), for investigationof the genome, and in miniaturizing technologies. Microscopy, cytometry,cell sorting, fluorescence correlation spectroscopy (FCS), ultra highthroughput screening (uHTS), multicolor fluorescence in situhybridization (mc-FISH), FRET-systems and microarrays (DNA- and proteinchips) are exemplary application fields. As known to one skilled in theart, a microarray, a grid-like arrangement where more than two differentmolecules are immobilized in a known predefined region on at least onesurface, is useful to evaluate receptor ligand interactions. As known toone skilled in the art, a receptor is a naturally occurring or syntheticmolecule that exhibits an affinity to a given ligand. Receptors can beused in a pure form or bound to another specie. Receptors can be coupledcovalently or noncovalently to a binding partner either directly orindirectly (e.g., through a coupling mediator). Receptor examplesinclude, but are not limited to, agonists and antagonists for cellmembrane receptors, toxins and other poisons, viral epitopes, hormonelike opiates and steroids, hormone receptors, peptides, enzymes, enzymesubstrates, drugs acting as cofactors, lectins, sugars,oligonucleotides, nucleic acids, oligosaccharides, cells, cellfragments, tissue fragments, proteins, antibodies, etc. As known to oneskilled in the art, a ligand is a molecule that is recognized by acertain receptor. Ligand examples include, but are not limited to,agonists and antagonists for cell membrane receptors, toxins and otherpoisons, viral epitopes, hormones like opiates and steroids, hormonereceptors, peptides, enzymes, enzyme substrates, drugs acting ascofactors, lectins, sugars, oligonucleotides, nucleic acids,oligosaccharides, proteins, antibodies, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows absorption/emission profiles of some inventive compoundsand commercial dyes.

FIG. 2 graphs functional assay results with some commercial dyes andinventive compounds with one conjugate produced in one embodiment.

FIG. 3 graphs functional assay results with some commercial dyes andinventive compounds with another conjugate produced in one embodiment.

FIG. 4 graphs functional assay results with some commercial dyes andinventive compounds with another conjugate produced in one embodiment.

FIG. 5 graphs functional assay results with some commercial dyes andinventive compounds with one conjugate produced in one embodiment.

FIG. 6 graphs functional assay results with some commercial dyes andinventive compounds with one conjugate produced in one embodiment.

FIG. 7 graphs functional assay results with some commercial dyes andinventive compounds with one conjugate produced in one embodiment.

FIG. 8 shows functional assay results with some commercial dyes andinventive compounds in one embodiment.

FIG. 9 graphs functional assay results with some commercial dyes andinventive compounds in one embodiment.

FIG. 10 graphs functional assay results with some commercial dyes andinventive compounds in one embodiment.

FIG. 11 tabulates functional assay results with some commercial dyes andinventive compounds in one embodiment.

FIG. 12 graphs functional assay results with some some commercial dyesand inventive compounds in one embodiment.

FIG. 13 tabulates functional assay results with some commercial dyes andinventive compounds in one embodiment.

FIG. 14 is a histogram showing functional assay results with somecommercial dyes and inventive compounds in one embodiment.

FIGS. 15A-D show immunofluorescence data with some commercial dyes andinventive compounds forming a conjugate in one embodiment.

FIGS. 16A-D show immunofluorescence data with some commercial dyes andinventive compounds forming a conjugate in one embodiment.

FIGS. 17A-D show immunofluorescence data with some commercial dyes andinventive compounds in one embodiment.

FIGS. 18A-D show immunofluorescence data with some commercial dyes andinventive compounds in one embodiment.

FIGS. 19A-C show immunofluorescence data with commercial dyes andinventive compounds in one embodiment.

FIG. 20A, column 1 A-E and column 2 A-E show immunofluorescence datawith commercial dyes and inventive compounds in one embodiment.

FIG. 20B, column 1 A-E and column 2 A-E show immunofluorescence datawith commercial dyes and inventive compounds in one embodiment.

FIG. 20C, column 1 A-E and column 2 A-E show immunofluorescence datawith commercial dyes and inventive compounds in one embodiment.

The following non-limiting examples further describe the compounds,methods, compositions, uses, and embodiments. They demonstrate that theinventive compounds exhibited desirable properties relative tocommercially available fluorescent dyes. Signal to noise ratio (S/N) isthe ratio between the desired signal and the mean of the blank,accounting for the standard deviation of the signal and the blank.Signal to background ratio (S/B) is the ratio between the desiredaverage signal and the average blank.

EXAMPLE 1 Synthesis of1,2-Dimethyl-1-(3-sulfopropyl)-1H-benzo[e]indole-6,8-disulfonic acid tripotassium salt

Five g (15.7 mmol) 6-hydrazino-naphthalene-1,3-disulfonic acid and 4.93g (25 mmol) 4-methyl-5-oxohexane sulfonic acid were dissolved in 50 mlacetic acid. The solution was heated at 140° C. for four hours. Thesolvent was evaporated in vacuum. The oily residue was dissolved in 20ml methanol, then 50 ml of a saturated solution of KOH in 2-propanolwere added to yield a yellow precipitate. The solid was filtered off anddried in vacuum. Yield 4.1 g, MS (ESI−): 158.2 [M]³⁻

EXAMPLE 2 Synthesis of3-(2-Methoxy-ethyl)-1,2-dimethyl-6,8-disulfo-1-(3-sulfopropyl)-1H-benzo[e]indolium

A mixture of 7.56 g (12.8 mmol)1,2-dimethyl-1-(3-sulfopropyl)-1H-benzo[e]indole-6,8-disulfonic acid tripotassium salt and 5.89 g (25.6 mmol) 2-methoxyethyl-p-toluene sulfonatewas heated under argon for 24 h. The residue was purified by columnchromatography (reversed phase silica, methanol/water, TFA). Yield 3.2g, MS (ESI−): 266.5 [M-2H]²⁻

EXAMPLE 3 Synthesis of3-(5-Carboxypentyl)-1,1,2-trimethyl-6,8-disulfo-1H-benzo[e]indolium

A mixture of 5.7 g (12.8 mmol)1,1,2-trimethyl-1H-benzo[e]indole-6,8-disulfonic acid dipotassium saltand 5 g (25.6 mmol) 6-bromohexanoic acid was heated under argon for 24h. The residue was purified by column chromatography (reversed phasesilica, methanol/water, TFA). Yield 1.4 g, MS (ESI−): 482.1 [M-H]⁻

EXAMPLE 4 Synthesis of3-(5-Carboxypentyl)-1,1-dimethyl-2-((1E,3E)-4-phenylamino-buta-1,3-dienyl)-6,8-disulfo-1H-benzo[e]indolium

0.97 g (2 mmol)3-(5-carboxypentyl)-1,1,2-trimethyl-6,8-disulfo-1H-benzo[e]indolium and0.57 g (2.2 mmol) malonaldehyde-bisphenylimine-hydrochlorid weredissolved in 10 ml acetic acid and 10 ml acetic anhydride and stirredfor four hours at 120° C. The solvent was removed under vacuum. Theresidue was washed carefully with ethyl acetate. A dark brown solid wasobtained which was processed without further purification. MS (ESI−):611.2 [M-H]⁻.

EXAMPLE 5 Synthesis of 679 Compound 1/1(2-{(1E,3E)-5-[3-(5-Carboxypentyl)-1,1-dimethyl-6,8-disulfo-1,3-dihydro-benzo[e]indol-(2E)-ylidene]-penta-1,3-dienyl}-3-(2-methoxy-ethyl)-1-methyl-6,8-disulfo-1-(3-sulfopropyl)-1H-benzo[e]indolium)

612 mg (1 mmol)3-(5-carboxypentyl)-1,1-dimethyl-2-((1E,3E)-4-phenylamino-buta-1,3-dienyl)-6,8-disulfo-1H-benzo[e]indoliumand 533 mg (1 mmol)3-(2-Methoxy-ethyl)-1,2-dimethyl-6,8-disulfo-1-(3-sulfopropyl)-1H-benzo[e]indoliumwere dissolved in 20 ml acetic acid/acetic anhydride (1/1) followed bythe addition of 200 mg of sodium acetate. The solution was stirred underreflux for 15 min. After cooling to room temperature, 20 ml diethyletherwas added. The resulting precipitate (mixture of the diastereomers 679Compound 1/1 (isomer 1) and 679 Compound 1/1 (isomer 2)) was extractedby suction, washed with ether, and dried.

The residue was purified by column chromatography (RP-18,acetonitrile/water and concentrated HCl) to separate the diastereomersfrom each other. The diastereomer that first eluted from the column wastermed diastereomer 1 (679 Compound 1/1 (isomer 1)). The diastereomerthat eluted second from the column was termed diastereomer 2 (679Compound 1/1 (isomer 2)). The diastereomers were separated, followed byneutralization and evaporation. Purification of the singlediastereomeric compound was completed on a RP-18 column,acetonitrile/water. The corresponding fractions were pooled and thesolvent was removed by distillation. The two products (diastereomers 679Compound 1/1 (isomer 1) and 679 Compound 1/1 (isomer 2)) were dried inhigh vacuum. 679 Compound 1/1 (isomer 1)

yield: 10%UV-vis (PBS): λ_(max) ⁼679 nm

-   -   λ_(em)=698 nm        MS (ESI−) [M/z]: 262.5 [M]⁴⁻; 357.7 [M+Na]³⁻        679 Compound 1/1 (isomer 2)        yield: 23%        UV-vis (PBS): λ_(max)=679 nm    -   λ_(em)=698 nm        MS (ESI−) [M/z]: 262.5 [M]⁴⁻; 357.7 [M+Na]³⁻

EXAMPLE 6

The following properties of 679 Compound 1-NHS were compared withcommercially available dyes.

679 Company B Cy5.5 DyLight DyLight Compound 1- Compound- Mono 680-NHS680B-NHS NHS NHS Ester MW (g/mol) 950 1196.16 1240.21 ~1150 1128.42 Ex(nm) 682 679 679 679 675 Em (nm) 715 698 698 702 694 ε 140,000 180,000180,000 184,000 250,000 (M−1cm−1) (theoretical)

Emission/excitation profiles for inventive and commercial compounds wasdetermined by reconstituting the compounds in DMF at 10 mg/ml and thendiluting in PBS buffer pH 7.2 to 10 μg/ml. Absorbance spectra werecollected on Cary UV spectrophotometer and emission spectra werecollected on Tecan Safire, shown in FIG. 1 where solid lines representabsorbance spectra and dashed lines represent emission spectra, forDyLight 680-NHS (purple), DyLight 680B-NHS (blue), 679 Compound 1-NHS(red), and Company B Compound-NHS (black). The maximum absorbance andemission for each compound is shown below.

Max Abs (nm) Max E nm) DyLight 680 677 715 DyLight 680B 680 704 679Compound 1 679 704 Company B 676 706 Compound

The following properties of 679 Compound 1-NHS, 679 Compound 4/4-NHS(V08-15173), and 679 Compound 4/4-NHS (V10-04152) were compared withcommercially available dyes.

Company B Company A DyLight 679 Compound V08-15173 V10-04152 Compound-Compound- 680B-NHS 1-NHS NHS NHS NHS NHS MW (g/mol) 1196.16 1240.211728.8 1524.75 ~1150 3241 Ex (nm) 679 679 684 689 679 681 Em (nm) 698698 706 721 702 698 ε 180,000 180,000 180,000 180,000 184,000 210,000(M⁻¹cm⁻¹) (theoretical) PEG 0 1/1 4/4 4/4 N/A ? (length/#of chain)Sulfonate 5 5 4 2 3 ?

EXAMPLE 7

Inventive and commercial compounds, each as the NHS ester, wereconjugated to goat anti-mouse (GAM) antibodies, goat anti-rabbit (GAR)antibodies, and streptavidin (SA). GAM, GAR, and SA, at a concentrationof 10 mg/ml in phosphate buffered saline (PBS), were dialyzed against 50mM borate buffer, pH 8.5. The compounds were reconstituted indimethylformamide (DMF) at 10 mg/ml and combined at 2X, 4X, 5X, 7.5X, or10X molar excess with GAM, GAR, or SA for two hours at room temperatureto label the antibodies or SA.

The labeled compounds, also termed dyes or labels, were subjected toPierce Dye Removal Resin (PDDR) to remove the unlabeled (free) compound;100 μl of the packed resin was used per mg of protein purified. Thepurified antibody-labeled dyes were then diluted 1:50 in PBS and scannedfor absorbance from 700 nm to 230 nm to determine the proteinconcentration, and to determine the mole dye to mole protein ratio. Eachconjugate was diluted 1:10 in 50% glycerol and heated in the presence of10 mM dithiothreitol (DTT) for 5 min at 95° C., then separated byelectrophoresis on polyacrylamide gels in the presence of sodium dodecylsulfate (SDS-PAGE). The gels were scanned using the Typhoon 9400 Imagerto verify removal of the unconjugated compound. Labeling efficiency wascompared, with results showing degree of labeling below.

DyLight DyLight 679 Company B 680 680B Compound 1 Compound Cy5.5 GAM-2X0.9 1.9 2.0 — 1.6 GAM-3X 1.3 2.7 3.0 — 2.2 GAM-4X 2.0, 1.9 3.6, 3.6 3.6,3.7 2.8 2.6 GAM-6X 2.3 5.0 5.1 3.9 — GAM-8X 2.4 5.6 5.0 4.3 — GAM-10X3.0 6.5 6.8 5.4 — GAM-12X 3.0 7.4 7.2 6.2 — GAR-1X 0.5 0.9 1.0 0.7GAR-2X 1.0 1.5 1.6 1.2 GAR-3X 1.3 2.1 2.4 1.7 GAR-4X 1.7, 1.8 2.6, 3.13.1, 3.1 2.3 2.2 GAR-6X 2.2 4.4 — — — GAR-8X 2.4 5.5 5.6 4.2 — GAR-10X3.5 6.0 6.7 5.3 — GAR-12X 3.4 6.6 6.7 6.2 — SA-6X 2.9 4.1 4.0 4.1 —SA-8X 3.4 4.7 5.0 4.8 —

Labeling efficiency of GAM, GAR and SA was equivalent for 679 compound1-NHS compared to DyLight 680B-NHS and higher compared to DyLight 680and Company B compound. The antibodies were labeled, purified, andevaluated by SDS-PAGE as described above. Antibodies were also labeledwith Company A compound, which was reconstituted in dimethylsulfoxide(DMSO) and combined at 2X, 5X, 7.5X, 10X, and 15X molar excess with GAMor GAR for sixty-five minutes at room temperature to label theantibodies.

Inventive and commercial compounds, each as the NHS ester, wereconjugated to goat anti-rat (GARat) antibodies. GARat, at aconcentration of 10 mg/ml in phosphate buffered saline (PBS), was spikedwith 10% v/v with 0.67 M borate buffer. The compounds were combined at5X or 10X molar excess with GAR at for 65 minutes at room temperature tolabel the antibody. The antibodies were labeled, purified and evaluatedby SDS-PAGE as described above.

Labeling efficiency of GARat was slightly higher for 679 Compound 1-NHScompared to DyLight 680B-NHS, V08-15173-NHS and Company B compound-NHS.

In another set of experiments, the labeling of GAM with the inventiveand commercial compounds is shown below.

Mole Mole Mole Mole Mole Dye/Mole Dye/Mole Dye/Mole Dye/Mole Dye/MoleProtein Protein Protein Protein Protein Ratio Ratio Ratio Ratio Ratio @2.5 X @ 5 X @ 7.5 X @ 10 X @ 15 X V08-15173 2.5 4.9 7.1 8.9 12.4V10-04152 2.8 5.1 7.4 10.0 13.9 DY679P1 1.8 4.1 6.6 8.5 12.9 Company A1.1 5.3 7.4 9.4 12.6 compound Company B 1.8 4.0 6.3 8.3 13.1 compound

Labeling efficiency of GAM was similar for all the dyes at all molarexcesses.

In another set of experiments, the labeling of GAR with the inventiveand commercial compounds is shown below, at a molar excess of 5X, 15X,and 25X.

Mole Mole Mole Dye/Mole Dye/Mole Dye/Mole Protein Protein Protein RatioRatio Ratio @ 5 X @ 15 X @ 25 X V08-15173 4.6 12.7 19.6 V10-04152 2.210.1 13.9 DY679P1 4.8 10.6 14.4 Company A 3.4 9.8 15.4 Compound CompanyB 4.1 15.2* 46.7* Compound

At 5X, the labeling efficiency of GAR for all the dyes was similarexcept for V10-04152. At 15X and 25X, the labeling efficiency wassimilar except for Company B compound. *Company B compound conjugatesprecipitated in the Slide-A-Lyzer at molar excesses greater than 10X,indicating this method of purification was not suitable for Company Bdyes.

EXAMPLE 8

Performance of the dye-GAM conjugates, dye-GAR conjugates, and dye-SAconjugates was evaluated in a functional assay. Wells of a 96 whiteopaque plate or black clear-bottom plate were coated with targetproteins mouse IgG immunoglobulin, rabbit IgG immunoglobulin, orbiotinylated bovine serum albumin (BBSA). One hundred μl mouse or rabbitIgG, or BBSA at a concentration of 10 μg/ml was applied to thecorresponding wells in columns 1 and 2. The target proteins wereserially diluted 1:1 from the wells in columns 2 to 11 using 100 μl PBS.One hundred μl of the samples from the wells in column 11 werediscarded. One hundred μl PBS was added to the wells in column 12. Theplates were incubated overnight at 4° C. and then blocked 2×200 μl withThermo Scientific SuperBlock® Blocking Buffer. The coated plates werewashed 2×200 μl with PBS-Tween and 1×200 μl with PBS. Based on thecalculated concentrations, conjugates were diluted 1:250 in PBS, addedto the corresponding plates (100 μl/well) and then incubated for onehour in the dark. The plates were washed with 2×200 μl with PBS-Tweenand 1×200 μl with PBS and filled with PBS buffer (100 μl/well) prior toscanning the white opaque plates on Tecan Safire using 679nm_(excitation)/702 nm_(emission) or scanning the black clear-bottomplates on LiCor Odyssey at 700 channel, to detect fluorescenceintensity.

As shown in FIGS. 2-7, RFU and/or signal to background ratio (S/B) ofthe dyes were compared at various concentrations, using the indicatedconjugation conditions.

FIG. 2 shows Tecan Safire results of a functional assay using GAMconjugated with either 8X molar excess of the dyes (solid lines) or 10Xmolar excess of the dyes (dashed lines) of DyLight 680 (purple filleddiamond/open square); DyLight 680B (blue filled triangle/X); 679Compound 1 (red filled circle/asterisk); and Company B Compound (blackfilled square/X sign). DyLight 680B-GAM (8X) showed higher bindingfluorescence compared to corresponding 679 Compound 1-GAM (8X). At 10Xmolar excess, 679 Compound 1-GAM showed similar performance to DyLight680B-GAM. Both DyLight 680 and Company B compound showed much lowerintensity compared to DyLight 680B and 679 Compound 1 conjugates.Signal/Background (S/B) was higher for DyLight 680B-GAM conjugates than679 Compound 1-GAM conjugates. Similar results were generally obtainedfor GAR conjugates.

FIG. 3 shows Tecan Safire results of a functional assay using SAconjugated with either 6X molar excess of the dyes (solid lines) or 8Xmolar excess of the dyes (dashed lines) of DyLight 680 (purple filleddiamond/open square); DyLight 680B (blue filled triangle/X); 679Compound 1 (red filled circle/asterisk); and Company B Compound (blackfilled square/+ sign). 679 Compound 1-SA (6X, 8X) showed slightly lowerbinding fluorescence compared to corresponding DyLight 680B-SA (6X, 8X).There was no quenching trend with the conjugates at higher molarexcesses. The S/B was slightly lower for 679 Compound 1-SA conjugatesthan for DyLight 680B-SA conjugates.

FIG. 4 shows LiCor Odyssey results of a functional assay using GAMconjugated with either 2X molar excess of the dyes (solid lines) or 4Xmolar excess of the dyes (dashed lines) of DyLight 680 (purple diamond);DyLight 680B (blue triangle); 679 Compound 1 (red circle); and Cy5.5(black asterisk). The following table compared S/B and raw intensitydata.

@ 1250 ng @ 39 ng @ 0 coating coating (blank) S/B DyLight 680-GAM-2X25.3 9.8 1.0 DyLight 680-GAM-4X 6.4 2.3 1.0 DyLight 680B-GAM-2X 18.2 4.61.0 DyLight 680B-GAM-4X 31.9 8.1 1.0 679 Compound 1-GAM-2X 36.1 7.9 1.0679 Compound 1-GAM-4X 65.2 15.9 1.0 Cy5.5-GAM-2X 62.3 17.7 1.0Cy5.5-GAM-4X 19.6 6.1 1.0 Raw Intensity DyLight 680-GAM-2X 1941300751514 76795 DyLight 680-GAM-4X 2596363 937396 403405 DyLight680B-GAM-2X 5179777 1311798 284501 DyLight 680B-GAM-4X 8614438 2190343270357 DY679P1-GAM-2X 5180336 1135001 143352 DY679P1-GAM-4X 74825101820225 114807 Cy5.5-GAM-2X 3957169 1124788 63513 Cy5.5-GAM-4X 43799011358039 223585

FIG. 5 shows LiCor Odyssey results of a functional assay using GARconjugated with either 2X molar excess of the dyes (solid lines) or 4Xmolar excess of the dyes (dashed lines) of DyLight 680 (purple diamond);DyLight 680B (blue triangle); 679 Compound 1 (red circle); and Cy5.5(black asterisk). The following table compared S/B and raw intensitydata.

@ 1250 ng @ 39 ng @ 0 coating coating (blank) S/B DyLight 680-GAR-2X66.7 19.8 1.0 DyLight 680-GAR-4X 62.2 17.4 1.0 DyLight 680B-GAR-2X 210.647.5 1.0 DyLight 680B-GAR-4X 167.2 39.3 1.0 DY679P1-GAR-2X 184.0 39.21.0 DY679P1-GAR-4X 141.9 33.4 1.0 Cy5.5-GAR-2X 57.3 16.1 1.0Cy5.5-GAR-4X 26.7 9.0 1.0 Raw Intensity DyLight 680-GAR-2X 1868227554324 28015 DyLight 680-GAR-4X 2140077 597726 34391 DyLight 680B-GAR-2X4178812 943008 19839 DyLight 680B-GAR-4X 6285439 1476347 37592DY679P1-GAR-2X 4039443 860737 21950 DY679P1-GAR-4X 6527381 1537020 46001Cy5.5-GAR-2X 2408459 675886 42049 Cy5.5-GAR-4X 2929556 988970 109613

FIG. 6 shows LiCor Odyssey results of a functional assay using GAMconjugated with either 8X molar excess of the dyes (solid lines) or 10Xmolar excess of the dyes (dashed lines) of DyLight 680 (purple diamond);DyLight 680B (blue square); 679 Compound 1 (red triangle); and Company BCompound (black X). The following table compared S/B data.

2500 ng mouse 39.1 ng mouse IgG/well IgG/well DyLight 680-GAM-8X 173.648.9 DyLight 680-GAM-10X 137.2 37.6 DyLight 680B-GAM-8X 929.3 142.4DyLight 680B-GAM-10X 914.5 165.6 DY679P1-GAM-8X 675.2 110.5DY679P1-GAM-10X 871.5 183.5 Company B Compound-GAM-8X 459.9 112.4Company B Compound -GAM-10X 374.0 96.1

679 Compound 1-GAM (10X) showed equivalent bound fluorescence comparedto corresponding DyLight 680B-GAM (10X). S/B was lower for 679 Compound1-GAM (8X, 10X) compared to DyLight 680B-GAM (8X, 10X). In general,similar results were obtained for GAR conjugates.

FIG. 7 shows LiCor Odyssey results of a functional assay using SAconjugated with either 6X molar excess of the dyes (solid lines) or 8Xmolar excess of the dyes (dashed lines) of DyLight 680 (purple diamond);DyLight 680B (blue square); 679 Compound 1 (red triangle); and Company BCompound (black X). The following table compared S/B data.

250 ng 3.9 ng BBSA/well BBSA/well DyLight 680-SA-6X 614.5 33.1 DyLight680-SA-8X 436.3 25.1 DyLight 680B-SA-6X 2196.7 43.8 DyLight 680B-SA-8X1969.5 75.6 DY679P1-SA-6X 1742.1 58.5 DY679P1-SA-8X 1406.0 54.7 CompanyB Compound-SA-6X 570.2 49.7 Company B Compound-SA-8X 427.7 36.1

679 Compound 1-SA (6X, 8X) showed lower bound fluorescence and S/Bcompared to corresponding DyLight 680B-SA (6X, 8X).

679 Compound 1 and Company Compound were conjugated to GAR at high molarexcesses, and evaluated in a functional assay, as described above. FIG.8 shows results expressed as RFU of a functional assay using GARconjugated with 679 Compound 1 at either a 7.5X molar excess (bluediamond), 15X molar excess (red square), or 22.5X molar excess (greentriangle), and Company A Compound at either 7.5X molar excess (purpleX), 15X molar excess (turquoise asterisk), or 22.5X molar excess (orangecircle).

FIG. 9 shows VarioSkan Flash results with excitation and emission of 679nm/702 nm, expressed as RFU of a functional assay using GAM conjugatedwith 5X molar excess of Company B compound (orange diamond), V08-15173(blue triangle), and V10-04152 (red circle). Based on the data,V08-15173-GAM (5X) showed higher binding fluorescence compared toCompany B Compound-GAM (5X).

FIG. 10 shows LiCOR Odyssey results, using the 700 channel, expressed asRaw Fluorescence Intensity of a functional assay using GAM conjugatedwith 5X molar excess of 679 Compound 1/1 (green triangle), Company ACompound (purple circle), and V08-15173 (blue square). FIG. 11 showsLiCOR Odyssey results expressed as S/B of a functional assay using GAMconjugated at 2.5X, 5X, 10X, or 15X molar excess of 679 Compound 1/1,Company A Compound, and V08-15173. V08-15173-GAM (5X) showed similarbinding fluorescence to Company A Compound-GAM (5X). 679 Compound1/1-GAM (5X) binding fluorescence was lower compared to the other twoconjugates.

FIG. 12 shows LiCOR Odyssey results, using the 700 channel, expressed asRaw Fluorescence Intensity of a functional assay using GAR conjugatedwith 5X and 25X molar excess of 679 Compound 1/1 (green 5X, diamond,solid line; green 25X, diamond, dashed line), Company A Compound (purple5X, triangle, solid line; purple 25X, triangle, dashed line), V08-15173(light blue 5X, square, solid line; dark blue 25X, square, dashed line),and V10-04152 (yellow 5X, circle, solid line; red 25X, circle, dashedline). FIG. 13 shows LiCOR Odyssey results expressed as S/B of afunctional assay using GAR conjugated at 5X, 15X, or 25X molar excess of679 Compound 1/1, Company A Compound, V08-15173, and V10-04152. Based onthe Raw Fluorescence Intensity data, there was no apparent quenching forthe V08-15173-GAR at 25X molar excess. 679 Compound 1/1, V10-04152, andCompany A Compound were saturating at 25X. FIG. 14 shows summary RawFluorescence Intensity data, and apparent quenching at 1000 ng/well at25X for 679 Compound 1/1 (679 1), V10-04152, and Company A Compound, butno apparent quenching at 25X for V08-15173.

EXAMPLE 9

The inventive compounds were evaluated for immunofluorescence in cellbased assays using the following protocol. Frozen A549 cell platesstored at −20° C. were placed for 30 min 50° C. Storage buffer (PBS) wasremoved and the cells were permeabilized for 15 min (100 μl/well) with0.1% Triton-X100 in 1×PBS buffer. Plates were blocked for 30 min in 2%BSA in 1×PBS-0.1% Trion-X100. Primary antibodies diluted in 2% BSA in1×PBS-0.1% Trion-X100 were added to the plates (column 1-11; column 12included only blocker) and incubated three hours at room temperature.Mouse anti-lamin A was added at 1 μg/ml and rabbit anti-lamin B1 wasadded at 3 μg/ml. After overnight incubation, the antibody solution wasremoved from the plates and the plates were washed with PBS-0.5%Tween-20 (2x 100 μl/well). GAM and GAR secondary antibodies labeled withDyLight 680-NHS, DyLight 680B-NHS, 679 Compound 1-NHS and Company BCompound-NHS were diluted to 4 μg/ml in PBS, added and incubated for onehour at room temperature. The plates were then washed three times with100 μl/well PBS, and Hoechst stain diluted to 0.1 μg/ml in PBS was addedto each well (100 μl/well). The plates were scanned on ArrayScan PlateReader for imaging and quantitation.

FIG. 15 shows results of an immunofluorescence assay using mouseanti-lamin A as a primary antibody, and DyLight 680-GAM (FIG. 15A),DyLight 680B-GAM (FIG. 15B), 679 Compound 1-GAM (FIG. 15C), or Company BCompound-GAM (FIG. 15D) as secondary antibody, where the compound wasconjugated to GAM (secondary antibody) at 4X molar excess (column 1), 6Xmolar excess (column 2), 8X molar excess (column 3), 10X molar excess(column 4), or 12X molar excess (column 5). 679 Compound 1 conjugated toGAM showed very similar performance to corresponding DyLight 680Bconjugates.

The inventive compounds and commercial dye were evaluated forimmunofluorescence in a second cell based assay using the followingprotocol. Frozen U20S cell plates stored at −20° C. were placedovernight at 4° C. Storage buffer (PBS) was removed and the cells werepermeabilized for 15 min (100 μl/well) with 0.1% Triton-X100 in 1×PBSbuffer. Plates were blocked for 30 min in 2% BSA in 1×PBS-0.1%Trion-X100. Primary antibodies diluted in 2% BSA in 1×PBS-0.1%Trion-X100 were added to the plates (column 1-11; column 12 includedonly blocker) and incubated five hours at room temperature. Mouseanti-lamin A was added at 2 μg/ml and rabbit anti-lamin B1 was added at4 μg/ml. After incubation, the antibody solution was removed from theplates and the plates were washed PBS-0.5% Tween-20 (2x 100 μl/well).Next, GAM and GAR secondary antibodies labeled with DyLight 680-NHS,DyLight 680B-NHS, 679 Compound 1-NHS, and Cy5.5 Mono Ester were dilutedto 4 μg/ml in PBS and incubated with the cells for one hour at roomtemperature. GAM and GAR conjugated to DyLight 680-NHS, DyLight680B-NHS, 679 Compound 1-NHS, and Company B Compound at 10X molar excesswere diluted to 4 μg/ml in PBS and then serially diluted 1:1 in theplate to the following concentrations: 2 μg/ml, 1 μg/ml, 0.5 μg/ml, 0.25μg/ml, 0.125 μg/ml and/or 0.0625 μg/ml. The plates were washed 3x with100 μl/well PBS, and Hoechst stain diluted to 0.1 μg/ml in PBS was addedto each well (100 μl/well). The plates were scanned on ArrayScan PlateReader for imaging and quantitation.

FIG. 16 shows results of an immunofluorescence assay using mouseanti-lamin A as a primary antibody, and either DyLight 680-GAM (FIG.16A), DyLight 680B-GAM (FIG. 16B), 679 Compound 1-GAM (FIG. 16C), orCy5.5-GAM (FIG. 16D) as secondary antibody, where the compound wasconjugated to GAM (secondary antibody) at 2X molar excess (column 1), 3Xmolar excess (column 2), or 4X molar excess (column 3). Performance inimmunofluorescence of 679 Compound 1 conjugate was similar to theperformance of corresponding DyLight 680B conjugates. Quantitativeanalysis of FIG. 16 data, expressed as Mean Total Intensity, which isthe average total intensity of all pixels within a defined area ordefined primary object such as a nucleus, is shown below.

Mean Total Intensity 2X 3X 4X DyLight 680 91899 122829 144792 DyLight680B 150542 228678 305700 DY679P1 232482 263580 330865 Cy5.5 211945342208 451111

Fluorescence signal intensity for DyLight 680B and 679 Compound 1 GAMconjugates was 2-4 times higher, depending on the molar excess, comparedto DyLight 680 or Cy5.5 GAM conjugates, and S/B for DyLight 680B & 679Compound 1 GAM conjugates at the low molar excesses was comparable toeach other and to DyLight 680. Overall fluorescence signal intensity forDyLight 680B and 679 Compound 1 GAR conjugates was about two timeshigher compared to DyLight 680. The S/B for DyLight 680B and 679Compound 1 GAR labeled at 2X, 3X or 4X molar excess were comparable toeach other and to DyLight 680 conjugates.

In the indicated experiments, the inventive compounds were evaluated forimmunofluorescence in cell based assays using the following protocol.Frozen A549 cell plates stored at −20° C. were placed for 30 min 50° C.Storage buffer (PBS) was removed and the cells were permeabilized for 15min (100 μl/well) with 0.1% Triton-X100 in 1×PBS buffer. Plates wereblocked for 30 min in 2% BSA in 1×PBS-0.1% Trion-X100. Primaryantibodies diluted in 2% BSA in 1×PBS-0.1% Trion-X100 were added to theplates (column 1-11; column 12 included only blocker) and incubatedthree hours at room temperature. Mouse anti-lamin A was added at 1 μg/mland rabbit anti-lamin B1 was added at 3 μg/ml. After overnightincubation, the antibody solution was removed from the plates and theplates were washed with PBS-0.5% Tween-20 (2×100 μl/well). GAM and GARsecondary antibodies labeled with DyLight 680-NHS, DyLight 680B-NHS, 679Compound 1-NHS and Company B Compound-NHS were diluted to 4 μg/ml inPBS, added and incubated for one hour at room temperature. The plateswere then washed three times with 100 μl/well PBS, and Hoechst staindiluted to 0.1 μg/ml in PBS was added to each well (100 μl/well). Theplates were scanned on ArrayScan Plate Reader for imaging andquantitation.

In the indicated experiments, the inventive compounds and commercial dyewere evaluated for immunofluorescence in a cell based assay using thefollowing protocol. Frozen U20S cell plates which were stored at −80° C.were thawed for 45 minutes at 50° C. Storage buffer (PBS) was removedand the cells were permeabilized for 15 minutes with 0.1% Triton-X100 in1×PBS buffer (100 μl/well). The cell plate was blocked for 60 minutes in2% BSA/PBS-0.1% Triton-X100. Primary antibody, either rat anti-Grp94 (5μg/ml), mouse anti-lamin A (10 tag/ml), or rabbit anti-lamin B1 (10μg/ml), diluted in 2% BSA/PBS-0.1% Triton-X100 was added to the plateand incubated for 1 hour at room temperature. Control wells containedonly 2% BSA/PBS-0.1% Triton-X100 blocker. After incubation, the antibodysolution was removed from the plate and the plate was washed three timeswith 100 μl/well of PBS-0.5% Tween-20 and one time with 100 μl/well PBS.GARat, GAM, or GAR secondary antibodies labeled with various molarexcess of the inventive or commercial compound were diluted to 4 μg/mlin PBS and incubated for 1 hour at room temperature. The plates werewashed three times with 100 μl/well of PBST and once with 100 μl/wellPBS, and Hoechst (diluted to 0.1 μg/ml in PBS) was added to each well(100 μl/well). The plates were scanned on ArrayScan Plate Reader orToxInsight Instrument.

FIG. 17 shows detection of Grp94 in U20S cells (column 1) with 679Compound 1-GARat (FIG. 17A), DyLight 680B-GARat (FIG. 17B),V08-15173-GARat (FIG. 17C), and Company B Compound-GARat (FIG. 17D)conjugated at a 5X molar excess; and associated controls (column 2).

FIG. 18 shows detection of Grp94 in U20S cells (column 1) with 679Compound 1-GARat (FIG. 18A), DyLight 680B-GARat (FIG. 18B),V08-15173-GARat (FIG. 18C), and Company B Compound-GARat (FIG. 18D)conjugated at a 10X molar excess; and associated controls (column 2).

As shown in FIGS. 17-18, no non-specific binding was observed withV08-15173-GARat and Compound B Compound-GARat conjugates but there waswith DY679P1 and DyLight 680B-GARat conjugates.

Quantitative analysis of the data of FIGS. 17-18, expressed as MeanTotal Intensity, which is the average total intensity of all pixelswithin a defined area or defined primary object such as a nucleus, isshown below.

Negative Controls S/B S/B 5X 10X 5X 10X (5X) (10X) 679 Compound 8122197162 36744 70585 2.2 1.4 1-GARat DyLight 680B-GARat 77855 91720 3477361355 2.2 1.5 V08-15173-GARat 76825 70881 28190 26341 2.7 2.7 Company B64881 56119 26762 30379 2.4 1.8 Compound-GARatS/B was slightly better for V08-15173-GARat conjugates (5X, 10X)compared to the corresponding 679 Compound 1-GARat, DyLight 680B-GARat,and Company B Compound-GARat conjugates.

679 Compound 1-GAM, Company A Compound-GAM, and Company A Compound R-GAMwere evaluated for immunofluorescence in a cell based assay usingdetection of PDI in cells with a mouse anti-PDI antibody. FIG. 19 showsresults of 679 Compound 1-GAM at a 15X molar excess (FIG. 19A), CompanyA Compound-GAM at a 15X molar excess (FIG. 19B), and Company A CompoundR-GAM at a 15X molar excess (FIG. 19C). As FIG. 19 shows, 679 Compound1-GAM exhibited proper staining of PDI in the ER while Company ACompound-GAM and Company A Compound R-GAM exhibited non-specificstaining with staining found throughout the cell. Similar results wereobtained for 679 Compound 1-GAM at 7.5X and 22.5X molar excesses, aswell as Company A Compound-GAM and Company A Compound R-GAM at 7.5Xmolar excess (data not shown).

FIG. 20A-C shows detection of lamin A in U20S cells (column 1) withV08-15173-GAM, V10-04152-GAM, 679 Compound 1/1-GAM, Company ACompound-GAM, and Company B Compound-GAM conjugates (4 μg/ml) at 5Xmolar excess (FIG. 20A), 10X molar excess (FIG. 20B), and 15X molarexcess (FIG. 20C) with V08-15173-GAM (row A FIGS. 20A, 20B, 20C),V10-04152-GAM (row B FIGS. 20A, 20B, 20C), 679 Compound 1/1-GAM (row CFIGS. 20A, 20B, and 20C), Company A Compound-GAM (row D FIGS. 20A, 20B,20C), Company B Compound-GAM (row E FIGS. 20A, 20B, 20C), and associatednegative controls (column 2). There was very little non-specific bindingobserved with V08-15173-GAM and 679 Compound 1/1-GAM. Company ACompound-GAM and Company B Compound-GAM conjugates showed highnon-specific binding starting from 5X molar excess. At low molar excess,Company A Compound-GAM exhibited staining of the nucleus, which wasgreater than the other dyes. V10-04152-GAM exhibited good specificitybut was not very bright. Staining of the nucleus with Company BCompound-GAM was greatly improved at 15X molar excess, but there was anincrease in non-specific binding.

The following table shows quantitative analysis of the FIGS. 20A-C dataexpressed as Mean Total Intensity, which is the average total intensityof all pixels within a defined area or defined primary object such as anucleus, and S/B ratios.

679 Compound Company A Company B V08-15173 V10-04152 1/1 CompoundCompound Negative Negative Negative Negative Negative Average controlcontrol control control control 2.5 X 17366 5966 11946 6085 29229 562854496 9107 7092 6329 5.0 X 32820 6643 12788 7357 49279 5859 72289 2030311922 8360 7.5 X 42876 7100 12513 6503 28782 152502 51104 18894 13351 10X 47756 9690 21147 6266 30098 6115 188505 136044 25020 18330 15 X 4906010779 15942 6746 48071 7277 679693 665370 67705 24350 679 CompoundCompany A Company B S/B V08-15173 V10-04152 1/1P1 Compound Compound 2.5X 2.9 2.0 5.2 6.0 1.1 5.0 X 4.9 1.7 8.4 3.6 1.4 7.5 X 6.0 1.9 4.9 3.01.4 10 X 4.9 3.4 4.9 1.4 1.4 15 X 4.6 2.4 6.6 1.0 2.8

Lamin A, a nuclear protein, should show staining specific to thenucleus. Any lamin A staining outside the nucleus is non-specificstaining. In addition, negative control conditions that lack a primaryantibody are also used to determine the antibody staining specificity.Company A and Company B Compounds showed no non-specific binding.V08-15173 and 679 Compound 1/1 exhibited a minimal amount ofnon-specific binding. Due to the strong non-specific binding at highermolar excesses, Company A and Company B Compounds exhibited decreasedsignal to background (S/B) levels.

Quantitative analysis of a repeat experiment of FIG. 20A-C are shownbelow.

V08-15173 Company A Compound 679 Compound 1/1 Negative Negative NegativeAverage control control control 2.5 X 118440 12053 198471 24758 771958711 5 X 183078 9441 351666 84270 140772 8674 10 X 391473 13094 638569330337 159948 20481 15 X 211270 17260 560119 632936 199626 16277 CompanyA S/B V08-15173 Compound 679 Compound 1/1 2.5 X 9.8 8.0 8.9 5 X 19.4 4.216.2 10 X 29.9 1.9 7.8 15 X 12.2 0.9 12.3

Company A Compound showed much higher non-specific binding compared toV08-15173 and 679 Compound 1/1, with the non specific binding appearingat the 2.5X condition for Company A Compound-GAM.

679 Compound 1/1-NHS and V08-15173-NHS showed on average a 20% lowerintensity compared to Company A Compound-NHS. V10-04152 intensity wasabout 50% lower than V08-15173, and about 65% lower than Company ACompound. GAM labeling efficiency was similar for all dyes at all molarexcesses. At 5X molar excess, the GAR labeling efficiency for all dyeswas similar, except for V10-04152. At 15X and 25X molar excesses, theGAR labeling efficiency similar for all compounds except Company BCompound. In immunofluorescence studies, Company A Compound, 679Compound 1/1, and Company B Compound GAM conjugates showed highnon-specific binding. V08-15173 and V10-04152 GAM conjugates showedlittle non-specific binding. Performance of conjugates inimmunofluorescence appeared to be highly dependent on the performance ofthe primary antibody.

EXAMPLE 10

The inventive compounds are used for in vivo imaging to obtaininformation about biological tissues that are primarily accessible. Thecompounds are responsive to light in the near infrared region of thespectrum, a part of the spectrum that has minimal interference from theabsorbance of biological materials. In one embodiment, the compounds areused for fluorescent imaging of targets within animals. For example, invivo imaging information can be obtained using methods such as X-ray,magnetic resonance imaging, positron emission tomography, ultrasoundimaging and probing, and other non-invasive methods used for diagnosingand treating disease. Light in the near infrared range (NIR), from about650 nm to about 1000 nm wavelength, can permeate through severalcentimeters of tissue and thus can be used for in vivo imaging.Fluorescent dyes, such as the inventive compounds that are responsive tolight in these longer wavelengths, can be used as conjugates withtargeting molecules such as antibodies to bind and accumulate in, e.g.,diseased tissue such as tumors, and may be used to distinguish healthyfrom diseased tissue. In some methods, the inventive compound may beattached to a biomolecule, such as a protein, peptide, or a drug, whichis localized or retained in the desired tissue environment. Fluorescentin vivo imaging using NIR dyes such as the inventive compounds arediagnostic agents to discretely target disease tissue directly withinanimals or humans.

For in-vivo imaging, the compound, an isomer of the compound, or aconjugate of the compound or isomer with a targeting agent, isadministered to a tissue (e.g., intravenously), permitted to accumulatewith excess compound removed by the circulatory system, then the tissueis irradiated with light at an appropriate wavelength. The NIRfluorescent light is recorded and/or an image is generated from the dataobtained to specifically detect and visualize the targeted cells ortissues. The dose of compound administered can differ and would be knownby one skilled in the art depending upon the specific tissue,application, etc., as long as the method achieves a detectableconcentration of the compound in the tissue to be assessed.

EXAMPLE 11 In Vivo Imaging Using an Inventive Compound Conjugated toAnti-HER2 Antibody

779 Compound 1-NHS is conjugated to a rabbit anti-HER2 antibody(Genscript USA, Piscataway N.J.) by reconstituting the compound indimethylformamide (DMF) at 10 mg/ml, then incubating at 10X molar excesswith rabbit anti-HER2 antibody (0.1 mg) for one hour at room temperatureto result in 779 Compound 1-anti-HER2 conjugate. The conjugationreaction is then subjected to PDDR to remove unlabeled (free) 779Compound 1. Ten microgram of conjugate is injected intravenously toathymic mice bearing BT474 tumors. The animals are imaged over time at1, 24, 48, 72, 96, and 120 hours post-injection using Pearl Imager,LI-COR Biosciences (LI-COR Instruments, Lincoln Nebr.).

Upon whole body imaging, fluorescence intensity is observed to bedistributed over the whole animal during the first hour imagining anddiminishes significantly at 72 hours. After 96 hours, the signal islocalized and specific to the tumor.

679 Compound 4/4-NHS is conjugated to a rabbit anti-HER2 antibody(Genscript USA, Piscataway N.J.) by reconstituting the compound indimethylformamide (DMF) at 10 mg/ml, then incubating at 10X molar excesswith rabbit anti-HER2 antibody (0.1 mg) for one hour at room temperatureto result in 679 Compound 4/4-anti-HER2 conjugate. The conjugationreaction is then subjected to PDDR to remove unlabeled (free) 679Compound 4/4. Ten microgram of conjugate is injected intravenously toathymic mice bearing BT474 tumors. The animals are imaged over time at1, 24, 48, 72, 96, and 120 hours post-injection using Pearl Imager,LI-COR Biosciences (LI-COR Instruments, Lincoln Nebr.).

Upon whole body imaging, fluorescence intensity is observed to bedistributed over the whole animal during the first hour imagining anddiminishes significantly at 72 hours. After 96 hours, the signal islocalized and specific to the tumor.

EXAMPLE 12 In Vivo Imaging Using Either Monosulfonated or DisulfonatedInventive Compound

The compound may be rendered less hydrophilic, i.e., more hydrophobic,by altering the number of sulfonate groups. The fewer sulfonates, themore hydrophobic the compound becomes. In this embodiment, the compoundmay be more readily retained in a desired tissue or location if theappropriate number of sulfonates is determined; e.g., compoundpenetration into cells is more efficient if fewer sulfonates are presenton the molecule. The compound may contain one, two, three, or foursulfonate groups. Hydrophobic compounds are also known to moreefficiently cross the cell membrane, and thus are more desirable whenthe target of interest is located within the cell.

Alendronate, a compound that binds to, and is retained in, LNCapprostate cancer cells, is conjugated with disulfonated or monosulfonatedbenzo 779 Compound 1 by incubating a solution containing 1 mMdisulfonated or monosulfonated 779 Compound 1-NHS in 1 ml of PBS and 0.5ml tetrahydrofuran (THF) with 0.1 mM alendronate and 0.2 mMdiisopropylethylamine at room temperature overnight. The conjugate ispurified using reverse phase HPLC with 0-50% methanol against a 0.1 Mammonium acetate buffer, and is then lyophilized.

LNCap cells are grown orthotopically in nude mice. 779 Compound1-alendronate (5 nmole) is injected into the tumor. Control mice areinjected with free 779 Compound 1 containing a carboxylic acid residueinstead of the reactive NHS ester. X-ray and near infra-red fluorescenceimages are captured.

Upon imaging the whole mouse, 779 Compound 1-alendroneate conjugate isretained in mouse tissue greater than the unconjugated compound; theconjugate is retained in the LNCap cell-induced tumor for at least 18hrs.

Alendronate, a compound that binds to, and is retained in, LNCapprostate cancer cells, is conjugated with disulfonated or monosulfonated679 Compound 4/4 by incubating a solution containing 1 mM disulfonatedor monosulfonated 679 Compound 4/4-NHS in 1 ml of PBS and 0.5 mltetrahydrofuran (THF) with 0.1 mM alendronate and 0.2 mMdiisopropylethylamine at room temperature overnight. The conjugate ispurified using reverse phase HPLC with 0-50% methanol against a 0.1 Mammonium acetate buffer, and is then lyophilized.

LNCap cells are grown orthotopically in nude mice. 679 Compound4/4-alendronate (5 nmole) is injected into the tumor. Control mice areinjected with free 679 Compound 4/4 containing a carboxylic acid residueinstead of the reactive NHS ester. X-ray and near infra-red fluorescenceimages are captured.

Upon imaging the whole mouse, 679 Compound 4/4-alendroneate conjugate isretained in mouse tissue greater than the unconjugated compound; theconjugate is retained in the LNCap cell-induced tumor for at least 18hrs.

EXAMPLE 13 In Vivo Imaging Using Either Monosulfonated or DisulfonatedInventive Compound

A drug delivery nanoparticle system conjugated with disulfonated ormonosulfonated 779 Compound 1 is prepared as follows. A solutioncontaining 1 mM disulfonated or monosulfonated 779 Compound 1-NHS in 1ml of PBS is incubated overnight at room temperature with 0.1 mM of ananti-cancer drug conjugated with transferrin in the form of ananoparticle. The resulting 779 Compound 1-nanoparticle conjugate ispurified by centrifugation and then lyophilized.

The 779 Compound 1 (isomer 1)-nanoparticle conjugate (1 nmole) isinjected intravenously into a mouse tail vein. Control mice are injectedwith free 779 Compound 1 dye. X-ray and near infra-red fluorescenceimages of mouse brain are captured.

779 Compound 1-nanoparticle conjugate localizes in the mouse brain forgreater than about 24 hours after injection. Tumor size progressivelydecreases after injection of 779 Compound 1-nanoparticle conjugate,compared to 779 Compound 1-nanoparticle without the anti-cancer drug.

A drug delivery nanoparticle system conjugated with disulfonated ormonosulfonated 679 Compound 4/4 is prepared as follows. A solutioncontaining 1 mM disulfonated or monosulfonated 679 Compound 4/4-NHS in 1ml of PBS is incubated overnight at room temperature with 0.1 mM of ananti-cancer drug conjugated with transferrin in the form of ananoparticle. The resulting 679 Compound 4/4-nanoparticle conjugate ispurified by centrifugation and then lyophilized.

The 679 Compound 4/4 (isomer 1)-nanoparticle conjugate (1 nmole) isinjected intravenously into a mouse tail vein. Control mice are injectedwith free 679 Compound 4/4 dye. X-ray and near infra-red fluorescenceimages of mouse brain are captured.

679 Compound 4/4-nanoparticle conjugate localizes in the mouse brain forgreater than about 24 hours after injection. Tumor size progressivelydecreases after injection of 679 Compound 4/4-nanoparticle conjugate,compared to 679 Compound 4/4-nanoparticle without the anti-cancer drug.

EXAMPLE 14

The mono-sulfonated derivative is on any one of eight possible positionson the 579, 679, or 779 compound, accounting for the stereochemistryaround the carbon positions on the rings as well as the non-symmetricalnature of the two ends of each dye. Similarly, the di- andtri-substituted sulfonates can be on multiple possible positions on theinventive compounds.

EXAMPLE 15

The inventive compounds are used for in vivo imaging as described in J.Gastrointest Surg (2008) 12:1938-1950. Briefly, human pancreatic celllines are maintained in media supplemented with penicillin/streptomycinat 37° C. with 5% CO₂. Mouse anti-CEA antibody and Control Mouse IgG (inPBS with 0.20% sodium azide) are conjugated to V08-15173. The dye isreconstituted at 10 mg/ml in DMF and then added to the antibody at a 10molar excess. The reaction is carried out for one hour at roomtemperature. The samples are then dialyzed against 3×2 L of PBS. Thecell lines are plated in 96-well plates at 5×10⁴ cells per well. After48 hours culture in appropriate media, the cells are incubated with 1 μgof V08-15173 labeled anti-CEA antibody or V08-15173-labeled controlmouse IgG for four hours at 37° C. The cells are then washed three timeswith PBS and then imaged with an inverted Nikon De-485 microscope andSpot camera RD.

Surgical procedures and intravital imaging are performed with theanimals anesthesized by intramuscular injection of 0.02 ml of 50%ketamine, 38% xylazine and 12 acepromazine maleate. Human pancreatic andcolorectal cancer cell lines are harvested by trypsinization and washedtwice with serum free medium and washed twice with serum-free medium.Cells (1×10⁶ in 100 μl of serum-free media) are injected subcutaneouslywithin 30 minutes of harvesting over the right flank in female nu/numice between 4-6 weeks of age. Subcutaneous tumors are allowed to growfor 7-14 days until they reached diameter of 1-2 mm prior to thedelivery of conjugated antibody. For ASPC-1 implants, the cells areharvested by trpsinization and washed 3X in serum-free media. The cellsare resuspended in serum-free media. The cells are resuspended inserum-free media at 5×10⁶/ml. A volume of 200 μl of the cell suspensionis then injected directly into the peritoneal cavity within 30 minutesof harvesting.

For antibody delivery, one to two weeks after subcutaneous, orthotopic,or intraperitoneal tumor implantation, animals are given intravenous(i.v.) injection of either conjugated anti-CEA or conjugated control IgGantibody diluted in PBS to a final volume of 100 μl. All i.v. injectionsare done via the tail vein. For the dose-response experiment, theantibody dose is 75 μg. For the in vivo time course, the animals areanesthesized and imaged at 30 min, 1, 2, 6, 24 hours and 8 and 15 daysafter systemic antibody delivery.

Predicted in vivo and ex vivo analysis results are that post-experimentsurgical exposure reveals accumulation of dye in the liver, bladder, anda region of inflammation in the subcutaneous tissue. Ex vivo analysis ofthe vital organs confirms the presence of dye predominantly in the liverwith some signal detected in the spleen intestines and lungs.

EXAMPLE 16

The inventive compounds are used for in vivo imaging as described in J.Gastrointest Surg (2008) 12:1938-1950. Briefly, human pancreatic celllines are maintained in media supplemented with penicillin/streptomycinat 37° C. with 5% CO₂. Mouse anti-CEA antibody and Control Mouse IgG (inPBS with 0.20% sodium azide) are conjugated to 779 Compound 1. The dyeis reconstituted at 10 mg/ml in DMF and then added to the antibody at a10 molar excess. The reaction is carried out for one hour at roomtemperature. The samples are then dialyzed against 3×2 L of PBS. Thecell lines are plated in 96-well plates at 5×10⁴ cells per well. After48 hours culture in appropriate media, the cells are incubated with 1 μgof V08-15173 labeled anti-CEA antibody or V08-15173-labeled controlmouse IgG for four hours at 37° C. The cells are then washed three timeswith PBS and then imaged with an inverted Nikon De-485 microscope andSpot camera RD.

Surgical procedures and intravital imaging are performed with theanimals anesthesized by intramuscular injection of 0.02 ml of 50%ketamine, 38% xylazine and 12 acepromazine maleate. Human pancreatic andcolorectal cancer cell lines are harvested by trypsinization and washedtwice with serum free medium and washed twice with serum-free medium.Cells (1×10⁶ in 100 μl of serum-free media) are injected subcutaneouslywithin 30 minutes of harvesting over the right flank in female nu/numice between 4-6 weeks of age. Subcutaneous tumors are allowed to growfor 7-14 days until they reached diameter of 1-2 mm prior to thedelivery of conjugated antibody. For ASPC-1 implants, the cells areharvested by trypsinization and washed 3X in serum-free media. The cellsare resuspended in serum-free media. The cells are resuspended inserum-free media at 5×10⁶/ml. A volume of 200 μl of the cell suspensionis then injected directly into the peritoneal cavity within 30 minutesof harvesting.

For antibody delivery, one to two weeks after subcutaneous, orthotopicor intraperitoneal tumor implantation, animals are given intravenous(i.v.) injection of either conjugated anti-CEA or conjugated control IgGantibody diluted in PBS to a final volume of 100 μl. All i.v. injectionsare done by tail vein. For the dose-response experiment, the antibodydose is 75 μg. For the in vivo time course, the animals are anesthesizedand imaged at 30 min, 1, 2, 6, 24 hours and 8 and 15 days after systemicantibody delivery.

Predicted in vivo and ex vivo analysis results are that post-experimentsurgical exposure reveals accumulation of dye in the liver, bladder, anda region of inflammation in the subcutaneous tissue. Ex vivo analysis ofthe vital organs confirms the presence of dye predominantly in the liverwith some signal detected in the spleen intestines and lungs.

The embodiments shown and described in the specification are onlyspecific embodiments of inventors who are skilled in the art and are notlimiting in any way. Therefore, various changes, modifications, oralterations to those embodiments may be made without departing from thespirit of the invention or the scope of the following claims. Thereferences cited are expressly incorporated by reference herein in theirentirety.

What is claimed is:
 1. A compound of

where each of R¹, R², R⁵, and R⁶ is the same or different and isselected from the group consisting of an aliphatic, heteroaliphatic,sulfoalkyl, heteroaliphatic with terminal SO₃, a PEG group P—Z where Pis selected from an ethylene glycol group, a diethylene glycol group,and a polyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, a sulfonamidegroup -L-SO₂NH—P—Z, and a caboxamide group -L-CONH—P—Z, where Z isselected from H, CH₃, a CH₃ group, an alkyl group, or a heteroalkylgroup; each of R⁷, R⁸, R¹¹, R¹², R¹³, and R¹⁴ is the same or differentand is selected from the group consisting of H, SO₃, a PEG group P—Zwhere P is selected from an ethylene glycol group, a diethylene glycolgroup, and a polyethylene glycol group, where the polyethylene glycolgroup is (CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, asulfonamide-containing group -L-SO₂NH—P—Z, and a carboxamide-containinggroup -L-CONH—P—Z, where Z is selected from H, a CH₃ group, an alkylgroup, or a heteroalkyl group; X is selected from the group consistingof —OH, —SH, —NH₂, —NH—NH₂, —F, —Cl, —Br, I, —NHS(hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O-STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, and—NR-L-NH—CO—CH2-I, where R is —H or an aliphatic or heteroaliphaticgroup; L is selected from the group consisting of a divalent linear(—(CH₂)_(t)—, t=0 to 15), crossed, or cyclic alkyl group optionallysubstituted by at least one oxygen atom and/or sulfur atom; Kat is anumber of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s) needed tocompensate the negative charge brought by the cyanine; m is an integerfrom 0 to 5 inclusive; o is an integer from 0 to 12 inclusive; and n isan integer from 1 to 3 inclusive.
 2. A compound selected from the groupconsisting of

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl, heteroaliphatic with terminal SO₃, a PEGgroup P—Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P—Z, and a caboxamide group-L-CONH—P—Z, where Z is selected from H, CH₃, a CH₃ group, an alkylgroup, or a heteroalkyl group; each of R⁷, R⁸, R¹¹, R¹², R¹³, and R¹⁴ isthe same or different and is independently selected from the groupconsisting of H, SO₃, a PEG group P—Z where P is selected from anethylene glycol group, a diethylene glycol group, and a polyethyleneglycol group, where the polyethylene glycol group is (CH₂CH₂O)_(s),where s is an integer from 3-6 inclusive, a sulfonamide-containing group-L-SO₂NH—P—Z, and a carboxamide-containing group —CONH—P—Z, where Z isselected from H, CH₃, a CH₃ group, an alkyl group, or a heteroalkylgroup; X is selected from —OH, —SH, —NH₂, —NH—NH₂, —F, —Cl, —Br, —I,—NHS (hydroxysuccinimidyl-/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O-STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,imidazole, azide, —O-carbodiimide, —NR-L-OH, —NR-L-O-phosphoramidite,—NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂, —NR-L-CO₂H, —NR-L-CO—NHS,—NR-L-CO-STP, —NR-L-CO-TFP, —NR-L-CO-benzotriazole, —NR-L-CHO,—NR-L-maleimide, —NR-L-NH—CO—CH₂—I, or —NR-L-NH—CO—CH₂—Br wherein R is—H or an aliphatic or heteroaliphatic group; L is selected from adivalent linear (—(CH₂)_(t)—, t=0 to 15), crossed, or cyclic alkyl groupoptionally substituted by at least one oxygen atom and/or sulfur atom;Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s) needed tocompensate the negative charge brought by the cyanine; m is an integerfrom 0 to 5 inclusive; o is an integer from 0 to 12 inclusive; each ofR3 and R4 is the same or different and is independently hydrogen, analiphatic group, a heteroaliphatic group, or a PEG group P—Z where P isselected from an ethylene glycol group, a diethylene glycol group, and apolyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, and Z isselected from H, CH₃, a CH₃ group, an alkyl group, or a heteroalkylgroup; or R3 and R4 together form a cyclic structure where R3 and R4 arejoined using a divalent structural element selected from the groupconsisting of —(CH₂)_(q)—, —(CH₂)_(q)O(CH₂)_(q′), —(CH₂)_(q)S(CH₂)_(q)—,—(CH₂)_(q)CH═CH—, —OCH═CH— where each of q and q′ is the same ordifferent and is a integer from 2 to 6 inclusive; and Y is selected fromthe group consisting of hydrogen, alkyl, sulfoalkyl, fluorine, chlorine,bromine, a substituted or unsubstituted aryl-, phenoxy-, phenylmercaptofunction, and a PEG group P—Z where P is selected from an ethyleneglycol group, a diethylene glycol group, and a polyethylene glycolgroup, where the polyethylene glycol group is (CH₂CH₂O)_(s), where s isan integer from 3-6 inclusive, and Z is selected from H, CH₃, a CH₃group, an alkyl group, or a heteroalkyl group.
 3. The compound of claim2 wherein each of R13 and R14 is sulfo.
 4. The compound of claim 2wherein each of R7, R8, R11, and R12 is sulfo. 5-8. (canceled)
 9. Amethod of labeling at least one biomolecule, the method comprisingproviding a composition comprising at least one excipient and thecompound of claim 2 in an effective concentration to label at least onebiomolecule under conditions sufficient for labeling the biomoleculewith the compound.
 10. The method of claim 9 wherein the biomolecule isselected from the group consisting of a protein, antibody, enzyme,nucleoside triphosphate, oligonucleotide, biotin, hapten, cofactor,lectin, antibody binding protein, carotenoid, carbohydrate, hormone,neurotransmitter, growth factors, toxin, biological cell, lipid,receptor binding drug, fluorescent proteins, organic polymer carriermaterial, inorganic polymeric carrier material, and combinationsthereof.
 11. (canceled)
 12. A method of labeling at least onebiomolecule, the method comprising providing a composition comprising atleast one excipient and a compound in an effective concentration tolabel at least one biomolecule under conditions sufficient for labelingthe biomolecule with the compound, where the compound is selected fromthe group consisting of

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl, heteroaliphatic with terminal SO₃, a PEGgroup P—Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P—Z, and a caboxamide group-L-CONH—P—Z, where Z is selected from H, CH₃, a CH₃ group, an alkylgroup, or a heteroalkyl group; each of R⁷, R⁸, R¹¹, R¹², R¹³, and R¹⁴ isthe same or different and is independently selected from the groupconsisting of H, SO₃, a PEG group P—Z where P is selected from anethylene glycol group, a diethylene glycol group, and a polyethyleneglycol group, where the polyethylene glycol group is (CH₂CH₂O)_(s),where s is an integer from 3-6 inclusive, a sulfonamide-containing group-L-SO₂NH—P—Z, and a carboxamide-containing group -L-CONH—P—Z, where Z isselected from H, CH₃, a CH₃ group, an alkyl group, or a heteroalkylgroup; X is selected from —OH, —SH, —NH₂, —NH—NH₂, —F, —Cl, —Br, —I,—NHS (hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O-STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,imidazole, azide, —O-carbodiimide, —NR-L-OH, —NR-L-O-phosphoramidite,—NR-L-SH, —NR-L-NH₂, —NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP,—NR-L-CO-TFP, —NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide,—NR-L-NH—CO—CH₂—I, or —NR-L-NH—CO—CH₂—Br wherein R is —H or an aliphaticor heteroaliphatic group; L is selected from a divalent linear(—(CH₂)_(t)—, t=0 to 15), crossed, or cyclic alkyl group optionallysubstituted by at least one oxygen atom and/or sulfur atom; Kat is anumber of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s) needed tocompensate the negative charge brought by the cyanine; m is an integerfrom 0 to 5 inclusive; p is an integer from 1 to 6 inclusive; each of R3and R4 is the same or different and is independently hydrogen, analiphatic group, a heteroaliphatic group, or a PEG group P—Z where P isselected from an ethylene glycol group, a diethylene glycol group, and apolyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, and Z isselected from H, a CH₃ group, an alkyl group, or a heteroalkyl group; orR3 and R4 together form a cyclic structure where R3 and R4 are joinedusing a divalent structural element selected from the group consistingof —(CH₂)_(q)—, —(CH₂)_(q)O(CH₂)_(q′)—, —(CH₂)_(q)S(CH₂)_(q′),—(CH₂)_(q)CH═CH—, —OCH═CH— where each of q and g′ is the same ordifferent and is a integer from 2 to 6 inclusive; and Y is selected fromthe group consisting of hydrogen, alkyl, sulfoalkyl, fluorine, chlorine,bromine, a substituted or unsubstituted aryl-, phenoxy-, phenylmercaptofunction, and a PEG group P—Z where P is selected from an ethyleneglycol group, a diethylene glycol group, and a polyethylene glycolgroup, where the polyethylene glycol group is (CH₂CH₂O)_(s), where s isan integer from 3-6 inclusive, and Z is selected from H, a CH₃ group, analkyl group, or a heteroalkyl group.
 13. The method of claim 12 whereinthe biomolecule is selected from the group consisting of a protein,antibody, enzyme, nucleoside triphosphate, oligonucleotide, biotin,hapten, cofactor, lectin, antibody binding protein, carotenoid,carbohydrate, hormone, neurotransmitter, growth factors, toxin,biological cell, lipid, receptor binding drug, fluorescent proteins,organic polymer carrier material, inorganic polymeric carrier material,and combinations thereof.
 14. The method of claim 12 wherein thecompound is V10-04152 or V08-15173.
 15. A method of detecting at leastone biomolecule, the method comprising providing a compositioncomprising at least one excipient and the compound of claim 2 in aneffective concentration to detect at least one biomolecule underconditions sufficient for binding the compound to the biomolecule, anddetecting the biomolecule-bound compound.
 16. The method of claim 15wherein the biomolecule is selected from a protein, antibody, enzyme,nucleoside triphosphate, oligonucleotide, biotin, hapten, cofactor,lectin, antibody binding protein, carotenoid, carbohydrate, hormone,neurotransmitter, growth factors, toxin, biological cell, lipid,receptor binding drug, fluorescent proteins, organic polymer carriermaterial, inorganic polymeric carrier material, and combinationsthereof.
 17. The method of claim 15 wherein the at least one biomoleculeis detected in an assay selected from fluorescence microscopy, flowcytometry, in vivo imaging, immunoassay, hybridization, chromatographicassay, electrophoretic assay, microwell plate based assay, fluorescenceresonance energy transfer (FRET) system, high throughput screening, ormicroarray.
 18. The method of claim 15 wherein the biomolecule isdetected by in vivo imaging comprising providing the biomolecule-boundcompound to at least one of a biological sample, tissue, or organism,and detecting the biomolecule within the at least one of a biologicalsample, tissue, or organism.
 19. (canceled)
 20. A method of detecting atleast one biomolecule, the method comprising providing a compositioncomprising at least one excipient and a compound in an effectiveconcentration to detect at least one biomolecule under conditionssufficient for binding the compound to the biomolecule, and detectingthe biomolecule-bound compound, where the compound is selected from thegroup consisting of

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl, heteroaliphatic with terminal SO₃, a PEGgroup P—Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a polyethylene glycol group, where thepolyethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P—Z, and a caboxamide group-L-CONH—P—Z, where Z is selected from H, CH₃, a CH₃ group, an alkylgroup, or a heteroalkyl group; each of R⁷, R⁸, R¹¹, R¹², R¹³, and R¹⁴ isthe same or different and is independently selected from the groupconsisting of H, SO₃, a PEG group P—Z where P is selected from anethylene glycol group, a diethylene glycol group, and a polyethyleneglycol group, where the polyethylene glycol group is (CH₂CH₂O)_(s),where s is an integer from 3-6 inclusive, a sulfonamide-containing group-L-SO₂NH—P—Z, and a carboxamide-containing group -L-CONH—P—Z, where Z isselected from H, CH₃, a CH₃ group, an alkyl group, or a heteroalkylgroup; X is selected from —OH, —SH, —NH₂, —NH—NH₂, —F, —Cl, —Br, —I,—NHS (hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O-STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,imidazole, azide, —O-carbodiimide, —NR-L-OH, —NR-L-O-phosphoramidite,—NR-L-SH, —NR-L-NH₂, —NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP,—NR-L-CO-TFP, —NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide,—NR-L-NH—CO—CH₂—I, or —NR-L-NH—CO—CH₂—Br wherein R is —H or an aliphaticor heteroaliphatic group; L is selected from a divalent linear(—(CH₂)_(t)—, t=0 to 15), crossed, or cyclic alkyl group optionallysubstituted by at least one oxygen atom and/or sulfur atom; Kat is anumber of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s) needed tocompensate the negative charge brought by the cyanine; m is an integerfrom 0 to 5 inclusive; p is an integer from 1 to 6 inclusive; each of R3and R4 is the same or different and is independently hydrogen, analiphatic group, a heteroaliphatic group, or a PEG group P—Z where P isselected from an ethylene glycol group, a diethylene glycol group, and apolyethylene glycol group, where the polyethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, and Z isselected from H, a CH₃ group, an alkyl group, or a heteroalkyl group; orR3 and R4 together form a cyclic structure where R3 and R4 are joinedusing a divalent structural element selected from the group consistingof —(CH₂)_(q)—, —(CH₂)_(q)O(CH₂)_(q′)—, —(CH₂)_(q)S(CH₂)_(q′),—(CH)_(q)CH═CH—, —OCH═CH— where each of q and g′ is the same ordifferent and is a integer from 2 to 6 inclusive; and Y is selected fromthe group consisting of hydrogen, alkyl, sulfoalkyl, fluorine, chlorine,bromine, a substituted or unsubstituted aryl-, phenoxy-, phenylmercaptofunction, and a PEG group P—Z where P is selected from an ethyleneglycol group, a diethylene glycol group, and a polyethylene glycolgroup, where the polyethylene glycol group is (CH₂CH₂O)_(s), where s isan integer from 3-6 inclusive, and Z is selected from H, a CH₃ group, analkyl group, or a heteroalkyl group.
 21. The method of claim 20 whereinthe biomolecule is selected from a protein, antibody, enzyme, nucleosidetriphosphate, oligonucleotide, biotin, hapten, cofactor, lectin,antibody binding protein, carotenoid, carbohydrate, hormone,neurotransmitter, growth factors, toxin, biological cell, lipid,receptor binding drug, fluorescent proteins, organic polymer carriermaterial, inorganic polymeric carrier material, and combinationsthereof.
 22. The method of claim 20 wherein the at least one biomoleculeis detected in an assay selected from fluorescence microscopy, flowcytometry, in vivo imaging, immunoassay, hybridization, chromatographicassay, electrophoretic assay, microwell plate based assay, fluorescenceresonance energy transfer (FRET) system, high throughput screening, ormicroarray.
 23. The method of claim 20 wherein the biomolecule isdetected by in vivo imaging comprising providing the biomolecule-boundcompound to at least one of a biological sample, tissue, or organism,and detecting the biomolecule within the at least one of a biologicalsample, tissue, or organism.
 24. The method of claim 20 wherein thecompound is V10-04152 or V08-15173.
 25. A kit for detecting at least onebiomolecule in a sample, the kit comprising the compound of claim 2 andat least one excipient, and instructions for use of the compound todetect a biomolecule in a sample. 26-28. (canceled)